Biochemical Techniques IT Lecture Notes 2023/2024 INTRODUCTION i, Standard biochemical techniques include: a. Spectrophotometry b. Chromatography cc. Enzyme kinetics dd, Ultra-centrifugation e. Isotope tracer techniques + Involves isolation, purification and characterization of proteins, nucleic acids and sub- cellular organelles * Biochemical techniques can be broadly grouped into: ‘+ Biochemical methods: purification of proteins nucleic acids, sub-cellular organelles, chromatography, metabolic labeling etc. ‘+ Immunological methods: immunoprecipitation, ELISA, Western blot technique, immunofluorescence staining ‘+ Molecular biology methods: PCR, DNA hybridization, RNA hybridization Protein and research techniques include: i, Chromatography Hope you have not forgotten these: ‘© Chromatography: defined as a process which affords the rapid and efficient analysis and separation of mixture of components due to their differential migration through a stationary phase under the influence of a moving phase. + Adsorption description: in this process, the components of a mixture in the liquid or gas phase, undergo selective adherence to the surface of a solid phase. The adsorbed substances are in turn selectively desorbed by suitable solvents. * Partioning: the components of a mixture undergo selective dissolution in immiscible solvent in which they have different relative solubilities.Principle © Column chromatography: operates on the principle that different components are adsorbed on the surface of an adsorbent to different extents depending upon their polarity and other structural features and thus get separated from each other * Chromatogram: when given sample's of mixture passed through the column, the constituents of mixtures get adsorbed on adsorbent to different extent, They form colored zones called chromatogram. ‘* Elution: the process of extraction of adsorbed compounds from adsorbent with the help of solvent is known as elution and the solvent is known as eluent. ‘* Elutropic Series: the arrangement of solvents in order of increasing eluting power. * Distribution Ratio: defined as concentration of solute in stationary phase to that in mobile phase. © Isocratic Elution: type of elution either one particular solvent of fixed composition is used as mobile phase or mixture of two or more solvents but ina fixed ratio is used as mobile phase * Gradient Elution: type of elution mixture of solvents with continuously increasing eluting power is used as mobile phase © Retention Time: the time of elution of the peak maximum is called retention time (tR) * Retention Volume: the volume of mobile phase required to eluate a component from the column. * Exclusion chromatography: chromatographic process in which the separation of the sample components takes place according to molecular size © MP solvent carrying a sample through the column (gas or liquid) + SP substance which stays fixed in a column and creates the separation (solid or viscous liquid) + The partitioning between liquid and solid support creates separation * Column chromatography: Gel filtratior/size exclusion chromatography ‘The separation technique based on the molecular size of the components. Separation is achieved by the differential exclusion from the pores of the packing material, of the sample molecules as they pass through a bed of porous particles. The principle feature of SEC is its gentle non-adsorptive interaction with the sample, enabling high retention of biomolecular activity. For the separation of biomolecules in aqueous or aqueous/ organic mobile phases, SEC is referred to as gel filtration chromatography (GFC), while the separation of organic 2ii, polymers in non-aqueous mobile phases is called gel permeation chromatography (GPC). NB: Here the large molecules leave the smaller molecules to stay in the stage longer Jon exchange Based upon the difference in exchange potentials of various ions. Here chromatography ion-exchange resin is used as stationary phase and during cation exchange, the cations with higher valency are retained and that with lower valency are removed. In cation exchange chromatography, positively charged molecules are attracted to a negatively charged solid support whereas in anion exchange chromatography, negatively charged molecules are attracted to a positively charged solid support. It is the most popular method for the purification of proteins and other charged molecules. Adsorption chromatography HPLC Involves the process of adsorption and desorption. Here, the stationary phase is solid on which the components get adsorbed. The mobile phase may be liquid or gas. If mobile phase is liquid it is called liquid-solid chromatography and if mobile phase is gas it is called gas solid chromatography and TLC is a special example of this type. Is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, itis forced through under high pressures of up to 400 atmospheres. That makes it much faster. It also allows you to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the ‘molecules flowing past it. This allows a much better separation of the components of the mixture. ‘Normal phase HPLC: is essentially the same as TLC. Although it is, described as "normal", it isn’t the most commonly used form of HPLC. Here, the column is filled with tiny silica particles, and the solvent is non- polar e.g, hexane. A typical column has an internal diameter of 4.6 mm or less and a length of 150 to 250 mm. Polar compounds in the mixture being passed through the column will stick longer to the polar silica than non- 3polar compounds will. The non-polar ones will therefore pass more quickly through the column. + Reversed phase HPLC: the column size is the same, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in them. A polar solvent is used - for example, a mixture of water and an alcohol such as ‘methanol. In this case, there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column, There will not be as much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent. Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules, for example. They therefore spend less time in solution in the solvent and this will slow them down on their way through the column. That means that now it is, the polar molecules that will travel through the column more quickly. Reversed phase HPLC is the most commonly used form of HPLC Chromatography can generally be classified as: 1. Classification based upon the Principle involved i. Partition chromatography i, Adsorption desorption chromatography Size Exclusion chromatography or Gel permeation chromatography iv. lon exchange chromatography 2. Classification based upon the type of equipment used i. Paper chromatography i, Thin layer chromatography iii, Column chromatography 3. Classification based upon the development pattern used + The process of separating the components of sample by passing mobile phase over the stationary phase is known as chromatographic development. It can be achieved by the following techniques: a) Elution Analysis: it is used for separation of two components of a mixture . The mixture is dissolved in a particular solvent, then put from the top on the adsorbent column. The different components move differently with the mobile phase through stationary phase depending upon their 4partitions coefficient and velocity of mobile phase. In adsorption-desorption chromatography separation of components occurs on the bases of differential adsorption of the components on the specific adsorbent. In partition chromatography separation occurs of difference in distribution of solute in the two phases. A plot of cone. of components in the element (mobile phase) as it comes out from the column is known as chromatogram. Elution is of two types Isocratic Elution: When mobile phase consists of a constant composition or the mobile phase may be a mixture of two or more liquids in fixed proportion, is called isocratic elution. Gradient Elution: In this different solvents in the increasing order of their eluting power or in the increasing order of their polarity are used as mobile phase b) Displacement Analysis: here, sample or mixture to be separated is introduced from the top in the form of thin layer and then eluent containing an active ingredient is transferred from the top which displaces the other components and push them forward in the column. This forms different zones in the decreasing order of distribution coefficients. Each zone displaces the next and all components are forced out of the column. Displacement method is advantageous as the sample is not diluted but here overlapping of zones occurs 50 cannot be used for quantitative analysis and after separation the column is loaded with the displacing agent whose removal is very difficult task. It is therefore, not a good method. ©) Frontal Analysis: here, the mixture to be separated is dissolved in mobile phase and added continuously from the top of the column. The components depending upon their adsorbing power gets separated on the stationary phase. The pure solvent moves ahead followed by less strongly adsorbed component and then more adsorbed component. Weakly adsorbed component comes on the front hence the name, frontal analysis. The first component will come out from the column at a constant composition until the column becomes saturated with second component. It has a disadvantage that large amount of mixture is required. 4. Classification based upon the nature of stationary and mobile phase used i ili iv. Liquid - Liquid chromatography Liquid - Solid chromatography Gas - Liquid chromatography Gas - solid chromatography Properties of chromatography matrix include: Rigidityii, Low non-specific intereaction iii, Chemical stability iv. Open pore structure Most commonly used packing materials for stationary phase (SP) * Cellulose * Crosslinked dextran (sephadex) + Agarose (sepharose) * Polyacrylamide Name" Type Tonizable group Remarks DEAB-clulose ‘Weakly basic Diethslaminoethyt Used to separate acidic nd =CH.CHNCH) neuteal proteins CMcatulose Weakly acidic Carboxymethy! Used to separate basic and neutral proteins P-elllose Strongly and weakly acide Disc: binds base proteins stronaly Bio-Rex 70 Weakly acid, Carboxylic aid Used to separate basic proteins polystyrene-based =cooH and amines DEAE Sephadex Weakly basic eros Diethylaminoethyt Combined chromatography and linked dextran ge =CH.CHNCH): ‘el itration of ace and neutral proteins ‘SP-Sepharose Strongly ace cross: Methyl sulfonate Combined chromatography and linked agarose el "H.SO.H ‘el filtration of basi proteins CM Bio-Gel A ‘Weakly acidic ers Carbosymethyt Combined chromatography and linked agarose gel =CH,COOH ge itration of basi and cute proteins ‘Sephadex and Sepharase pls are manufactured by Amersham Paanmaca Hotsh, Pasta, New Jere: Hin Rex ein and Bio-Gel are mano {acted by oR Laborer, Heres, Calon, Detection of proteins and methods used include: i, Electrophoresis ii, Enzyme-linked immunosorbent assay Western blot iv. Immunochemistry Biochemical methods for nucleic acid research i, Nucleic acid purification ii, Molecular cloningiii, DNA sequencing iv. Polymerase chain reactions a. Reverse transcription polymerase chain reaction b._ Real time polymerase chain reaction v. Detection of nucleotides a. Souther blotting b._Insitu hybridization Protein Extraction, Isolation, Quantification and Purification ‘+ Cell lysis is the first step in cell fractionation, organelle isolation and protein extraction and purification Call lysis opens the door to a myriad of proteomics research methods Many techniques have been used to obtain the best possible yield and purity for different species of organisms, sample types (cells or tissue) and target molecule or subcellular structure. Structure and Diversity of Cells Allcells have a plasma membrane. Membrane proteins are embedded in the lipid bilayer, held in place by one or more domains spanning the hydrophobic core. * Peripheral proteins bind the inner or outer surface of the bilayer through interactions with integral membrane proteins or with polar lipid head groups. The nature of the lipid and protein content varies with cell type and species of organism. ‘+ nanimal cells, the plasma membrane is the only barrier separating cell contents, from the environment, but in plants and bacteria the plasma membrane is also surrounded by a rigid cell wall. ‘+ Bacterial cell walls are composed of peptidoglycan. Yeast cell walls are composed of two layers of &-glucan, the inner layer being insoluble to alkaline conditions, Both of these are surrounded by an outer glycoprotein layer rich in the carbohydrate mannan, Plant cell walls consist of multiple layers of cellulose. These types of extracellular barrier confer shape and rigidity to the cells. ‘+ Plant cell walls are particularly strong, making them very difficult to disrupt mechanically or chemically. Until recently, efficient lysis of yeast cells required. mechanical disruption using glass beads, whereas bacterial cell walls are the easiest to break compared to these other cell types. The lack of an extracellular wall in animal cells makes them relatively easy to lyse.Cell Lysis and Protein Extraction Historically, physical lysis was the method of choice for cell disruption and extraction of cellular contents These methods often require expensive, cumbersome equipment and involve protocols that can be difficult to repeat due to variability in the apparatus. The traditional physical disruption methods are not conducive for high throughput and smaller volumes typical of modern laboratory research, Currently, detergent-based lysis methods have become widely common. Different detergent-based solutions composed of particular types and concentrations of detergents, buffers, salts and reducing agents have been developed to provide the best results for particular species and types of cells. Detergents have both lysing and solubilizing effects, Cell Fractionation and Organelle Isolation ‘+ By careful optimization of physical disruption techniques, detergent-buffer solutions and density gradient methods, procedures have been developed to enable separation of subcellular structures or classes of compounds. Combination of tools and steps enable isolated and study of intact nuclei, mitochondria and other organelles. Protease Inhibition and Protein Stabilization * Cell lysis disturbs the carefully controlled cellular environment, allowing endogenous proteases and phosphatases to become unregulated. As a result extracted proteins become degracled or artifactually modified by the activities of these molecules. ‘© To prevent these effects and obtain the best possible protein yield in cell lysis, protease and phosphatase inhibitors are added to the lysis reagents. Numerous compounds have been identified and used to inactivate or block the activities of proteases and phosphatases by reversibly or irreversibly binding to them. © When the goal of cell lysis is to purify or test the function of a particular protein, special attention must be given to the effects of the lysis reagents on the stability and function of the protein(s) of interest. Certain detergents will inactivate the function of particular enzymes, and long-term stability of extracted/ purified proteins often requires that they be removed from the initial lysis reagents and/or stabilized by addition of particular compounds. Protein Refolding ‘+ Some cell lysis and protein solubilization methods cause the denaturation of proteins. Functional tests with such proteins require that they be renatured. Many proteins spontaneously refold into their native, functional structures 8when the denaturing solubilization reagents are removed by purification methods such dialysis. Other proteins, however, will fold into non-functional and even insoluble forms by this process. In such cases, specialized sets of buffer conditions must be tested to identify those that promote the highest possible yield of properly refolded protein.METHODS OF PROTEIN PURIFICATION AND ANALYSIS Separation, identification, and characterization of proteins is based on their different properties and is essential for the analysis of the complex mixtures of proteins found in cells or tissues * Purification of a protein is an essential step in elucidating its structure and function, Purification involves the fractionation or separation of proteins from each other in mixture and evaluation of their level of purity following each step of purification used. Typically, to completely purify a protein from all others in a complex mixture requires a number of steps, Purified = homogeneous (starting from cell extract the protein mixture is heterogeneous) Strategy of protein purification select a source break open cells, separate components keep the protein native (usually cold) develop an assay to follow the protein run the purification steps based on the properties of the protein Properties that are used to separate proteins: molecular weight or size solubility and stability charge binding affinity hydrophobicity Reasons for purifying proteins detailed studies on function determination of structure industrial pharmaceutical applications generate antibodies amino acid sequence determination paose Methods of Stabilization of protein 1. Changing buffer + dialysis + ii ultracentrifugation 2. Concentrating protein + helps to maintain active protein «sometimes addition of a carrier protein may help + addition of glycerol 3. Inhibitors 10+ iLproteases phosphatase inhibitors 1. Methods for separating on the basis of size + Electrophoresis using SDS-PAGE + Gel filtration chromatography and dialysis or ultrafiltration + Ultracentrifugation 2. Methods for separating on the basis of solubility + Ammonium sulfate precipitation or fractionation + Solvent solubility © Differential solubility of proteins in solvents of different dielectric constants 3. Methods for separating on the basis of charge + Ton exchange chromatography * Isoelectric focusing 4, Methods for structural determinations of proteins lid + X-ray diffraction (crystallography) © 3D structure determination + NMR spectroscopy ~ liquid © provides structural and functional information about particular atoms in a protein + Circular dichroism spectroscopy — amount and type of secondary structure + Mass spectroscopy — determination of very small amounts of protein. Proteins are typically identified and characterized by the following methods: a. Ultracentrifugation b. Spectroscopy ©. Chromatography 4. Electrophoresis e. Enzyme-linked methods ‘Two other general approaches for separation of proteins also include: i, Dialysis/ultrafilration + Dialysis © Protein solution is placed in a cylinder of cellophane tubing sealed at one end Tubing is then sealed at the other end and suspended in a large volume of buffer © Cellophane is semipermeable, so proteins remain inside the tubing while low- molecular-weight solutes diffuse out and are replaced by solutes of the buffer aii, © Pores allow small molecules, such as those of solvents, salts, and small metabolites, to diffuse across the membrane but block the passage of larger molecules © Solvent in which proteins are dissolved is exchanged by dialysis for a buffer solution suitable for subsequent technique © Ammonium sulfate fractionation -- requires dialysis of precipitate or soluble fraction. Ammonium sulfate, (NH4)2SO4 , is the most commonly used salt for salting out proteins because its large solubility in water, its relative freedom from temperature effects, and it has no harmful effects on most of the proteins, its availability and cost © Dialysis is one of the common operations in biochemistry to separate dissolved molecules by passing through a semi-permeable membrane according to their molecular dimensions. © Cellophane (cellulose acetate) is the most commonly used dialysis material although many other substances such as nitrocellulose and collodion are available. © Atequilibrium the concentration of small molecules outside and inside the bag is the same while the macromolecules remain inside the bag © Three factors affect the rate of dialysis: + the concentration differences of that molecules between the internal and external solution (which is the driving force for the movement of the molecules) + mixing on both sides of dialysis membrane will increase the rate of ‘movement prevent the small particles on the side of low concentration. + dialyzable particles size versus pore size of the membrane, substances, that are very much smaller than the pore size will reach equilibrium, faster than substances that are only slightly smaller than the pores. Ultrafiltration © Similar principle except cellophane or membrane used as a filter through which material is spun + Eluate small enongh to pass through + Retentate - too large to pass through pores © Membranes with pore sizes ranging from a MW of 1000 to 1,000,000 are available Ammonium sulfate precipitation or fractionation Salting out of proteins based on differences in neutralization from salt to make protein insoluble due to lack of interaction or solvation with water Enough ammonium sulfate is mixed with a solution of proteins to precipitate the less soluble impurities, which are removed by centrifugation. Target protein and other, more soluble proteins remain in the fluid, called the supernatant fraction. More ammonium sulfate is added to the supernatant fraction, until the desired protein is precipitated. Mixture is centrifuged, the fluid removed, and the precipitate dissolved in a minimal volume of butfer solution. 2Ammonium sulfate gives a two- to threefold purification (that is, one-half to two-thirds of the unwanted proteins have been removed from the resulting enriched protein fraction). Solubility of Proteins in salt solution 1 act ),80, ° 80, su] Lo | 0 10 20 30 40 lon strength At low ionic strength, increased salt increases solubility (salting- in) Increased salt concentrations causes the protein to precipitate out of solution (salting out) NB This occurs due to competition between the added salt with other dissolved solutes for solvation Polyethyleneimine (PEI) precipitation PE! is a positively charged molecule in solutions of neutral pH. It binds to negatively charged macromolecules such as nucleic acid and acidic proteins and forms a network of PEI and bound acidic molecules that rapidly precipitates. The binding is stoichiometric A heavy precipitate rapidly forms that can be pelleted by centrifugation Whether an acidic protein binds to PEI depends on the salt concentration. At low salt (0.1 M NaCl), a mildly acidic protein will bind and precipitate, but at intermediate salt (0.4 M NaCl), it will elute from the polymer and become soluble. A highly acidic protein will bind at low salt, not be solubilized at intermediate salt, and will be eluted at high salt (0.9 M NaCl) ‘There are three different strategies for the use of PEI precipitation: © Strategy A: Precipitate with PEI at high salt (IMNaCl). This precipitates the nucleic acids and leaves almost all protein in the supernatant. B© Strategy B ( for neutral or basic proteins): Precipitate with PEI at 0.1 M NaCl to remove nucleic acids and acidic proteins. This leaves protein of interest in the supernatant, © Strategy C ( for acidic proteins such as E. coli RNA polymerase) Other precipitation methods include: Ethanol and acetone precipitation + Precipitation with organic solvents, such as ethanol and acetone, has been in use for well over a hundred years, but is probably best known for its use in fractionating human serum in the classic work of Cohen and Edsall. Care must be taken to carry out precipitations at very cold temperatures to avoid protein denaturation. Isoelectric precipitation * Proteins are less soluble at their isoelectric point where they have zero net charge and can most easily approach each other with minimal charge repulsion. Since proteins are also less soluble at very low ionic strength, isoelectric precipitation is usually done at very low or no salt. Thermal precipitation + Inthis method, cell extracts are heated to a temperature at which many proteins denature and precipitate, where the protein of interest is more stable and stays soluble. This approach is particularly useful in purifying enzymes from thermophiles expressed in E. coli where the extract is heated to a high enough temperature, often to denature and precipitate almost all E. coli protein, leaving the heat stable enzyme in solution. 14SEDIMENTATION * Sedimentation of suspended and other dissolved particles occurs due to centrifugal force * Sedimentation is based on two major principles i Separates solicl matter as a pellet from dissolved solutes as a supernatant ji, Separates soluble macromolecules of different mass or density Centrifugation © Acentrifuge is an equipment used for separation of macromolecules such as: i. cells fi, subcellular components iii, proteins iv. nucleic acids The separation is based on: i size fi, shape iii, density iv. viscosity of the medium v. speed of the rotor + NB: The method utilizes the density difference between particles and the medium in which they are dispersed. + Dispersed systems are subjected to artificially induced gravitational fields (centrifugation) ‘© Substances whose densities are higher than that of solvent will sink (sediment) and vice versa co. The greater the particles are in density, the faster they will move (sediment) © Whenever there is no difference in density (isopycnic condition), the particle will stay steady © acentrifuge provides a much greater force (centrifugal force) to separate particles which exhibit tiny differences in density © acentrifuge gives artificially induced gravitational fields to sediment particles faster. Composition of centrifuge i. acentrifuge comprises of: i. anelectric motor to pass electricity through the coil, ie. converts electricity into (angular) motion ii, a drive shaft to transmit mechanical power iii, a rotor to hold tubes ji, a centrifuge is also used to provide centrifugal force to drive other processes such as ultrafiltration Types of Centrifuge iii, Classification of Centrifuges depend on: 15i, maximum speed of sedimentation and an example is the Microfuge, 10,0003, ii, presence/Absence of vacuum and an example is the Ultracentrifuge, 600,000g iii, temperature control refrigeration and an example is the High Speed refrigerated centrifuge, 100,000, iv. volume of samples and capacity of centrifugation tubes and an example is the Large-capacity preparative centrifuge, 3,000-7,000g iv. Another classification of centrifuges is based on the design and type of rotor used: i. fixed angle rotor ii, swinging bucket rotor Fixed Angle Rotor « supernatant ERS Sedimenting particles have only Longer distance of travel may allow short distance to travel before better separation, such as in density pelleting. Shorter run time. gradient centrifugation. Easier to The most widely used rotor type. _ Withdraw supernatant without disturbing pellet. Density gradient sedimentation Sedimentation through density gradient overcomes the following: © peletting of small particles in the bottom of the tube due to: + mechanical vibration ‘thermal gradients * convection currents ‘Types of density gradient centrifugation + First sedimenting particles will be contaminated with slow sedimenting ones. + Mechanical vibrations, thermal gradient, convection currents also affect sedimentation properties of a molecule. + Tominimize the above problems, centrifugation through a dense gradient is used 161. Rate Zonal Centrifugation = common technique for macromolecules separation, density of the solvent 2. Isopyenic (equilibrium) centrifugation where the solvent density encompasses density of the particle. + Samples are overlaid onto a pre-formed gradient (sucrose, CsCl, Ficoll, hypaque, Percoll etc) and then centrifuged. + Separation is based on the sedimentation coetffiecient (s value) of the molecule. + Svalue is determined by the molecular weight, density, size and shape of the molecule. + The selection of the correct centrifugation time is vital (too short a time is not enough to separate the molecule and too long a spin leads to peletting of one or more species) Ultracentrifugation + Molecular Weight and Shape = fundamental physical properties of a protein. + Estimates of molecular weight can be obtained using SDS-PAGE or gel filtration + One very useful technique for measuring molecular weight and shape is centrifugation. + A particle that's subjected to a centrifugal field by being spun in a centrifuge is subjected toa force, f=m(-+pjarr where m is the mass of the particle, ris the distance of the particle from the center of, rotation, and @ is the angular velocity. @-¥P) ~ poyancy factor, which accounts for the fact that particle, is buoyed up by the surrounding solvent. Vis the specific volume of the particle (ml/g) (= 1/ density of the particle). + Movement of particle through the solvent is resisted by a frictional coefficient, f that depends on the shape of the particle. Frictional coefficient is an important factor in any transport process, such as centrifugation or gel filtration. ‘A spherical particle has f= 1.0, whereas a cigar-shaped or cylindrically-shaped particle will have f> 1.0. + Movement of any particle under the influence of a centrifugal field is characterized by its sedimentation coefficient, 8, which is directly proportional to its molecular mass, M, and inversely proportional tof, 7M(1-vp) S TT where Nis Avogado's number. + Ultracentrifugation is used in two ways to characterize proteins: In sedimentation equilibrium experiments, In sedimentation velocity experiments, the the centrifuge is operated at a relative low centrifuge is operated at maximal speed, speed so that the forces of sedimentation and which causes the protein to sediment to the diffusion balance and the protein distributes bottom of the tube. The rate at which the in the centrifuge cell ina manner boundary moves gives 8, which when proportional to its molecular weight. combined with M gives f, a measure of the shape of the protein. + Spinning around an axis creates a centrifugal field + A particle within a centrifugal field experiences a centrifugal force (Fe = mor) + Fe= the centrifugal force, m = mass of the particle, w = angular velocity and r = distance from the axis + Centrifugal force is opposed by: buoyant and frictional forces which can be defined as: F, = -mo'r and F, = -fv Where m, = the mass of the displaced solution, f = frictional coefficient and v = velocity of the particle. The particle will move at a velocity such that the total force equals 0, thus: FL+F, +P, = 0, or mo’r - mo'r - fy = 0 substituting mop, mg, where v = partial specific volume of the particle and p, = density of the solvent, and solving for v results in: v= a'rm(1 - vp) /f£ = o'rmo(p, - p)/f Conclusions: sedimentation 1 The more massive a particle, the faster it moves in a centrifugal field 2. The densera particle (ie, the smaller its v) the faster in moves in a centrifugal field 183. The denser the solution, the slower the particle will move in a centrifuugal field. 4. The greater the frictional coefficient (factors such as viscosity, particle shape, ete influence this parameter), the slower the particle will move, 5. The particle velocity is 0 when the solution density is greater than the particle density | force (wr) the faster the particle sediments. 6 The greater the centrifi Forces interacting in centrifugation iedimenting force, m,*r, is opposed by... Roemce m, = the mass of equal volume of solvent = om | Frictional Resistance against Feces particle moving through fluid. fiction + Fruoyancy =fv f = frictional coefficient of particle in the solvent v = particle velocity . Flotation Force F=m, roo? BALANCE between the sedmenting force and counteracting force Net force = (M,, —m,)ro? - fv 19Sedimentation coefficient Movement of any particle under the influence of a centrifugal field is characterized by its sedimentation coefficient, 8, which is directly proportional to its molecular mass, M, and inversely proportional tof. Movement of particle through the solvent is resisted by a frictional coefficient, f, that depends on the shape of the particle. When the frictional force balances the driving force, z : it (m,-m, -fv =0 n(l-tp) f where S = terminal velocity / unit acceleration w Sedimentation coefficients have units of sec. 10-4 sec is called 1 svedberg (or | S). T, Svedberg pioneered research on sedimentation in an ultracentrifuge. 20Uses of centrifugation in the laboratory; i. Remove cellular debris from blood to separate ell fre plasma or serum ii. Concentrate cellular elements and other components for microscopic analysis or chemical analysis, fi, Separate protein bound ar antibody bound ligand from free ligand in immunological assay. iv, Extract solutes from aqueous or organic solvents. ve Separate lipid components like chylomicrons from other components of plasma, ELECTROPHORESIS Electrophoresis = separation of charged molecules by their movement or migration in an electric field (typically in or on a solid support) — separation can be based on their net charge or on the basis of their mass if they can be made to have the same charge to mass ratio. + Polyacrylamide (PAGE = polyacrylamide gel electrophoresis) © Most common matrix + Other matrices include agarose (more typical for nucleic acids or very large proteins) © Protein samples are placed in a highly cross-linked gel matrix, and an electric field is applied © Matrix is buffered to a mildly alkaline pH so that most proteins are anionic and migrate toward the anode (What if protein is positively charged at pH 8.52) © Gel matrix retards the migration of large molecules more than smaller molecules as they move in the electric field © Proteins are thus fractionated on the basis of both charge and mass © Cross-linking of monomers and linking agents can be adjusted to control level of porosity of the matrix + This allows a matrix to be optimized for the separation of a particular size of proteins + SDS-PAGE (Sodium dodecyl sulfate) © Modification of the standard electrophoresis technique which uses the negatively charged detergent sodium dodecyl sulfate (SDS) to overwhelm the native charge on proteins so that they are separated on the basis of mass only © SDS detergent is added to the matrix as well as to the protein samples © Samples are heated with SDS in presence of a reducing agent (2-mercaptoethanol or dithiothreitol) to reduce any disulfide bonds and thus break those covalent bonds + This treatment denatures the protein thus breaking secondary, tertiary, and quarternary interactions © Dodecyl sulfate anion, which has a long hydrophobic tail, binds to hydrophobic side chains of amino acid residues in the polypeptide chain + SDS binds at a ratio of approximately one molecule for every two residues of a typical protein. Since larger proteins bind proportionately 21more SDS, the charge-to-mass ratios of all treated proteins are approximately the same. © SDS-protein complexes are highly negatively charged and move toward the anode ‘© Rate of migration through the gel is inversely proportional to the logarithm of their mass, + Larger proteins encounter more resistance and therefore migrate more slowly than smaller proteins + Sieving effect differs from gel-filtration chromatography + Gel filtration = larger molecules are excluded from the pores of the gel and hence travel faster + SDS-PAGE = all molecules penetrate the "pores" of the gel, so the largest proteins travel most slowly © Protein bands that result from this differential migration can be visualized by staining + Stains = coomassie blue or silver stain (much more sensitive) + Autoradiography can also be used if the protein is radioactive © Molecular weights of unknown proteins can be estimated by comparing their migrations to the migrations of reference proteins electrophoresed on the same gel. ‘SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is used to assess the purity and to estimate the molecular weight of a protein ‘The stacking Gel 22sd Ly ee — re a — r 1-— —_ r 0 web 0 — r 5-— — r 5-— be — 0 Gel Stacking Gel Interactions: ‘y. when an electrical current is applied to gel, ions carry the current to the anode (+). vi. Cl- ions, having the highest charge/mass ratio migrate faster, being depleted at cathode end and ‘concentrated at anocle end. vi. glycine from electrophoresis buffer enters gel at pH 6.8 and becomes primarily zwitterionic ‘moving slowly. (pKal=2.5, pKa2=9.6 and pl-6.0) viii protein, coated with SDS has a higher charge/mass ratio than glycine so moves fast, but slower than Cr. ix. when protein encounters resolving gel it slows down due to increased frictional resistance (smaller pore size), allowing following protein to “catch up” or stack Xs protein is depleted from cathode end, glycine must carry current so begins to migrate behind protein, in essence concentrating the proteins further at stacking gel resolving gel interface. 2Resolving Gel Interactions: + when glycine reaches resolving gel it becomes anionic and migrates much faster than protein due to higher charge/mass ratio. + now proteins are sole carrier of current and separate according to their molecular mass due to sieving effect of pores in gel 2D-PAGE high resolution + Isoelectric focusing ~separates proteins on the basis of their isoelectric points © Support medium is a gel in which a pH gradient has been established (ampholytes) © Protein applied migrates through gel until it reaches its pl or where it has no net charge and thus can no longer move in the electric field © Since each protein has its own distinct pl (based on AA composition and structure) protein mixtures can be separated based on their pl + 2: gels = combination of SDS-PAGE and isoelectric focusing © Isoelectric focusing in first dimension and SDS PAGE in second dimension © Examples of a 2-D gels + Silver stained human liver protein 2D-PAGE picture obtained with non linear immobilised pH gradient (IPG) from 35 to 10 for the first dimensional separation + Silver stained plasma protein 2D-PAGE picture obtained with non linear immobilized pH gradient (IPG) from 3.5 to 10 for the first dimensional separation, 0.75 L or 45 gm of plasmatic proteins were loaded on the gel. + 2D PAGE provides the highest resolution for protein analysis and is an important technique in proteomic research, where resolution of thousands of proteins on a single gel is sometimes necessary le S0S-PAGE First dimension, Second dimension, tube gel or strip gel slab gol ‘xi, Protein samples prepared for SDS-PAGE analysis are denatured by heating in the presence of sample buffer containing 19 SDS with or without a reducing agent such as 20mM DTT, 2- ‘mercaptoethanol (BME) ot TCEP. The protein sample is mixed with the sample buffer and boiled for 3 to 5 minutes, then cooled to room temperature before it is pipetted into the sample well of a gel 24F, Antibodies * Antibodies (Ab) are proteins with high specificity for a particular antigen (foreign substance) that can be used to identify, quantitate, and localize specific proteins + Use of antibody shows high specificity for a particular protein in contrast to other methods described here which are general methods * Monoclonal vs. polyclonal Ab + Western blots using SDS- + ELISA and RIA + Insitu localization using immunoflourescence Spectroscopy Spectroscopy = quantitation and general identification of the presence of protein + Absorption spectroscopy © entities peptide bonds and certain functional groups in proteins by measuring the absorbance of different wavelengths by a solution using either visible or ultraviolet light + Peptide bonds absorb UV light in the range of 180-200 nm ‘+ Aromatic rings of Phe, Trp, and Tyr absorb UY light in the range of 260-300 rm with a general absorbance maximum at ~ 280 nm + This is often used to get a rough estimate of protein quantity or concentration in solution during purification + Note that denaturing of proteins alters their secondary or tertiary structure and leads to changes in absorption at 180-230 nm + Flourescence spectroscopy © More sensitive measurement than UV or visible spectroscopy in detecting the presence and quantity of protein © Flourescence occurs when molecule in an excited state (produced by exciting the molecule with a high energy, short wavelength of light) re-emits some of its excess energy in the form of a longer wavelength of light (excitation vs. emission wavelengths) Only proteins with aromatic side chains flouresce © Proteins can be made to flouresce by coupling a flourescent tag to the protein + Colorimetric determinations (reactions that produce color that can be read by spectroscopy) © Asolution of proteins is treated with a reagent that leads to the binding of a dye specifically to proteins © Amount of dye or color bound formed is proportional to protein content in the solution ‘The absorbance of the solution with dyes bound to the protein is read in a spectrophotometer o Examples of assays or reagents: Biuret, Lowry, Bradford, BCA Ultracentrifugation © Ultracentrifugation = protein separation on the basis of size 25© Sedimentation coefficient of a protein subjected to a centrifugal force (outward) depends on the protein's mass, density, shape and on the density of the suspending, solution + Suspending solution often a gradient of sucrose from 5-40% ‘Svedberg unit (S) is a measure of the sedimentation coeftecient of a particle (10'! see) © Magnitude of the coefficient gives a relative value that can characterize the molecular weight of a protein + Rate zonal or band centrifugation + Examples of sedimentation coefficients of molecules, particles, cells Chromatography Chromatography = color picture (literal meaning from observing color separation of leaf pigments) Paper separation - separation on paper with different solvents Column separation - most common today + Column chromatography © acylindrical column is filled with an insoluble material such as substituted cellulose fibers or synthetic beads, which is called the matrix © the protein mixture is applied to the column and washed through the matrix by the addition of solvent. © as solvent flows through the column, the eluate (the liquid emerging from the bottom of the column) is collected in many fractions © the rate at which proteins travel through the matrix depends on interactions between matrix and protein. For a given column, different proteins are eluted at different rates, © the concentration of protein in each fraction can be determined by measuring the absorbance of the eluate at a wavelength of 280 nm. (Recall that at neutral pH, tyrosine and tryptophan absorb UV light at 280 nm.) © to locate the protein of interest, the fractions containing protein must then be assayed, or tested, for biological activity or some other characteristic property of the protein. + thus, you must know some property or characteristic of the protein you are interested so that you can identify it in the fractions you produce Assay for the Protein General Types of Chromatography + Gel filtration (size exclusion) — separates proteins on the basis of molecular size © Utilizes a matrix of porous beads + Proteins that are smaller than the average pore size of the beads enter and penetrate the matrix filling much of the internal volume of the beads and are therefore retarded as they move through the beads. 26+ Note that a protein will thus enter and migrate through many of the beads (the number depends on the size of the protein compared to the porosity of the beads) and thus its flow through the column will be relatively slow + Incontrast, molecules that are larger than the pores are excluded and rapidly flow through the column + The following generalities thus can be stated: + The smaller the protein, the later it elutes from the column, + Fewer of the pores are accessible to the larger protein molecules. Consequently, the largest proteins flow past the beads and elute first. + Examples of Gel Filtration Media + Sephadex, Sepharose, Bio-Gel common brand names + Come in a variety of different pore sizes Ton exchange -- matrix carries positive charges (anion-exchange resins) or negative charges (cation-exchange resins)(Stryer Fig. 3-9) © Anion-exchange matrices bind negatively charged proteins, retaining them on the matrix for subsequent elution (DEAE — diethylaminoethyl) © Conversely, cation-exchange materials bind positively charged proteins (CM - carboxymethyl) © Proteins bound by charge to the matrix can be serially eluted by gradually increasing the salt concentration in the solvent passed over the column. + Like-charged salt ions and proteins bind to the matrix competitively. The presence of high enough concentrations of like charged ion will result in competition for binding to the matrix and bound proteins will be "competed off" + How effectively a protein can be "competed off is dependent on the strength of the interaction of a protein with the matrix which isa distinct property of each protein (its amino acids composition and structure) © One common matrix to which these groups are attached is cellulose Affinity ~ relies on binding interactions between a protein and a ligand © Most selective type of column chromatography © Aligand is a molecule, group, ion, or atom that binds~-usually noncovalently--to another molecule or atom © Inaffinity chromatography, the ligand is covalently attached to the matrix. + Itmay be a substance to which an enzyme binds in vivo (substrate), or it may be an antibody that recognizes the protein of interest © Asa mixture of proteins passes through the column, only the target protein specifically binds to the matrix based on "affinity" © The column is then washed with a mild buffer several times to rid the column of any nonspecifically bound proteins The target protein is then eluted by washing the column with a solvent containing a high concentration of the free ligand, or perhaps a concentrated, 27solution of salt (disrupts interaction between protein and ligand) = compete off bound protein Affinity chromatography alone can sometimes purify a protein 1000- to 10,000- fold ina single purification step 28Overview and an example of Protein (enzyme) purification strategy The objectives in enzyme or protein purifications or preparations are: ‘© tomaximize the yield, ‘© tomaximize the purity ‘+ tomaximize the specific activity (the ratio of catalytic activity to total protein amount, i.e. a measure of the homogeneity of the preparation) s to keep in mind are that; ‘+ enzymes are usually relatively unstable and have to be treated gently ie. to minimize temperature-dependent processes such as protein degradation and denaturation (unfolding). ‘+ proteins in cells also occur in the presence of other biochemicals, other enzymes and proteins, many of which often have very similar physical and/or chemical properties, which makes their separation difficult. ‘+ these molecules may also stabilize the protein and may be required for the activity of the protein Typically one tries to find a good source of the desired enzyme, which really means a source in which the enzyme is present in a high relatively concentration e.g. myoglobin in whale muscle. © Oncea crude solution of the enzyme is obtained it is usually first subjected to separation methods based on solubility to initially increase the concentration of the desired enzyme and decrease the concentration of other protein impurities. To study a particular protein in the laboratory, one must usually separate it from all other cell components, including other similar proteins. + Never forget that crude mixtures of cellular proteins may contain thousands of different proteins! An overview of the more commonly used methods in protein purification in the order they are typically used: Choice of cell source Choice of cell extracts Removal of small molecules Removal of nucleic acids, Heat treatment Fractionation by precipitation Fractionation by gel adsorption Ton exchange chromatography Gel filtration Preparative electrophoresis Affinity Chromatography ‘+ Determination of homogeneity: electrophoresis, isoelectric focusing. ‘The first step in protein purification is to prepare a solution of proteins. 29The source of a protein is often whole cells or tissues in which the target protein accounts for less than 0.1% of the total dry weight. ‘+ Isolation of an intracellular protein requires that cells or chopped tissue be suspended in a buffer solution and homogenized i.e. disrupted into cell fragments ‘+ Most proteins will dissolve in the buffer solution. Assume that the desired protein is one of many proteins in this solution, Next step in protein purification is to carry out several sequential different separations or fractionations on the cellular extracts. + These methods include ‘+ Change in pH (solubility minimum at pl, pH denaturation of impurities). © Change in ionic strength (salting in or out)(ammonium sulfate fractionation followed by dialysis) + Decrease in dielectric constant (addition of organic solvents). + Heat denaturation (unfolded contaminating proteins may aggregate). ‘+The partially purified material is then usually subjected to one ‘or more chromatographic steps, e.g. © Gel filtration (based on size) © Tonexchange (based on charge) © Affinity chromatography (based on specific binding to a covalently linked ligand bound to the column, support-e. g. a His-tag) © Absorption chromatography (e.g. celite, hydroxyapatite or phosphocellulose) © Sometimes preparative isoelectric focusing or chromatofocusing is used as the last stage of the + Atvarious stages along the way techniques such as dialysis, ultrafiltration (to remove high salt concentrations or other low molecular weight material), and lyophilization (freeze drying, to remove water) will be used. + Criteria for measuring homogeneity include; © electrophoresis (native and SDS) © isoelectric focusing © N-terminal amino acid analysis. + The specific activity serves as a relative measure of the enzyme's purity, + Note: © Itis vital to keep a table indicating the volumes, yields, total amount of enzyme and protein and specific activity for each step of the isolation procedure. 30Example of Purification Scheme for Penicillinase from a Bacterium Penicillinase (or -lactamase) hydrolyzes the -lactam bond of penicillin antibiotics and is responsible for much of the resistance to penicillin antibiotics today. The enzyme is produced extracellularly or in the periplasm, either naturally or from a cloned gene. The bacteria are grown till the yield is maximum, the cells then spun down ina centrifuge (and if necessary ruptured osmotically). The supernatant is then mixed with celite (a type of clay which is very absorbent) or an ion-exchange resin, which absorbs the penicillinase. This is allowed to settle or spun down in a centrifuge. The enzyme is eluted with a high salt concentration (about 2M), then dialyzed to remove the salt. The resulting solution is loaded onto a cation (carboxymethyl cellulose) chromatography column and eluted with a gradual salt gradient at pH 7. The desired fraction, containing the -lactamase is located by a combination of A280 and assays for catalytic activity. These fractions are pooled, dialyzed to remove excess salt, and subjected to preparative isoelectric focusing. The product is homogeneous by SDS PAGE (polyacrylamide gel electrophoresis) and analytical isoelectric focusing gels. Summary + The aims of any protein purification process include: i, removal of unwanted contaminants ii, concentration of desired protein iii, tun protein into a form ready for intended application iv. transfer protein into a stable environment ‘+ Anassay is the biochemical test that characterizes a molecule + Anassay can be quantitative or semi-quantitative (qualitative) but either way, is important to determine the presence and quantity of a biomolecule ‘+ Most biomolecules occur in minute amounts in the cell, and their detection and analysis require the biochemist to first assume the major task of purifying them from amongst many thousands of molecules found in extracts + Purification procedures are almost as diverse as the diversity of biomolecules ‘+ The methods for purification of bimolecular range from simple precipitation, centrifugation, and gel electrophoresis to sophisticated chromatographic and affinity techniques that are constantly undergoing development and improvement. * Chromatography techniques are sensitive and effective in separating and concentrating, minute components of a mixture (widely used for quantitative and qualitative analysis in medicine, industrial processes etc) ‘+ Besides ultracentrifugation and gel electrophoresis, chromatography is one of the methods used to determine the molecular weight of biomolecules, since it has the a1highest resolution in the purification of native biomolecules and is valuable when both the purity and the activity of a molecule are of importance The methods are based on such properties as size and shape, net charge and bioproperties of the biomolecules studied Proteins can be purified by various methods, mainly chromatographic, given * a source of the protein + a detection method specific for the protein of interest Progress of purification can be followed to monitor: i, total protein (need a method to measure protein concentration) ii, total activity (or other specific property) of protein of interest ili, Specific activity (total activity /total protein), which indicates relative degree of purity; constant specific activity is one indication that protein may be pure. Separation methods based on: i. differential centrifugation (to separate soluble from particulate fractions of crude cell lysate) ii, solubility (fractional precipitation, "salting out") ‘The main protein separation/purification methods are: i. column chromatography ii, gel filtration (also called size exclusion chromatogram; molecular sieve chromatogram.), based on size and shape iii, ion exchange chromatography (based on net charge of protein at the working pH) iv. _ affinity chromatography (based on specific ligand binding) Protein Extraction and Purification studies on pure proteins are essential for understanding structural and functional properties of proteins the goal of protein purification is to separate the protein you want from other proteins and small molecules mild conditions to avoid denaturation (usually low temperature, 0-4° C, and avoiding extremes of pH) need detection method (e.g. biological activity, or spectroscopy) usually use several purification methods, one after another start with (mixture of) proteins in buffered solution, e.g. extract of proteins from cells that have been lysed (broken open) Protein Purification i Source of Protein to purify a protein one needs a source i.e blood or some other biological fluid, but most often whole cells, usually a specific type (liver, muscle, yeast, bacteria, etc.) cells lysis by osmotic shock or by mechanical disruption such as with a tissue homogenizer to disrupt cell membranes to release proteins in soluble form without damaging the protein membrane-bound proteins can also be purified, but different approaches are required 32ii, Fractionation of homogenate ‘+ usually by differential centrifugation ie produces several fractions (successive pellets, supernatants) of decreasing density, each with lots of proteins NB: Assay each fraction to find which one contains most of the protein of interest, and fractionate that further by more discriminating methods such as chromatography iii, Detection Method (Assay) '* Detection methods are based on differences in: i. properties of the protein(s) ii, differential solubility iii, size iv. charge v. __ binding affinity for specific ligands, etc ‘The method used must be able to: i. measure the specific protein of interest in order to separate it from other contaminating molecules in the mixture ii, test for unique property of protein of interest, e.g, specific catalytic activity of an enzyme, or spectroscopic property of a unique prosthetic group iii, measure total amount of protein present in the mixture by different methods such as colorimetric measurements or absorbance of protein (e.g., A280nm, which really is detecting aromatic sidechains) Steps of Protein extraction Initial fractionation of homogenate ‘© usually by differential centrifugation --> several fractions (successive pellets, supernatants) of decreasing density, each with lots of proteins ‘+ assay each fraction to find which fraction contains most of the protein of interest, and fractionate that further by more discriminating methods. Separation Methods * fractional precipitation (based on differences in solubility properties) : salting out often the first step in purification + proteins require HO molecules interacting with surface groups, in order to stay in aqueous solution (hydration) + salting out usually uses increasing concentrations of ammonium sulfate [(NH4)2SO4] to compete with the protein groups for the available HzO * method is crude (no precise separations) but a good way to rapidly get rid of a lot of impurities * like all purification methods, salt fractionation has to be worked out empirically for each protein of interest + NB: Every protein in the solution has its own solubility limits in ammonium sulfate, independent of the other proteins in the mixture. 33Solubility is affected by; © Concentration of the protein of interest, o pH © temperature © But generally, i small proteins are more soluble than large proteins ji, the larger the number of charged side chains, the more soluble the protein iii, proteins are usually least soluble at their isoelectric points (determined by protein's amino acid composition) iv. solubility of proteins depends on the surface properties of each individual type of protein, NB: One of the most useful method for concentrating a protein is to precipitate it out and then redissolve it in smaller volume; sounds simple, isn’t it? Separation Method Property 1. Precipitation ‘Ammonium sulfate Solubility Polyethylene glycol Solubility 2. Chromatography on-exchange (anion or cation) Net charge Hydrophobic interaction Surface hydrophobicity Metal affinity Metal-binding sites Ligand affinity Ligand-binding sites (e.g. NAD, NADP) Gel filtration Subuniv/oligomer size, shape 3. Centrifugation Size, shape 34RADIOIMMUNOASSAYS Radioimmunoassay (RIA) is a binding assay in which the binder is an antibody (*) which uses radioactivity to measure amount of bound of free antigen. The radioactively labeled antigen is called tracer. RIA is a very sensitive technique used to measure concentrations of antigens without the need to use a bioassay. In this method either the antigen or antibody is labeled with radioisotope. However, the method is costly and requires special precautions due to use of radioactive materials Ithas therefore, been largely replaced by enzyme linked immunosorbent assay (ELISA) which uses colorimetric signals rather than radioisotopes Name suggests 3 components (antibody, solid support/sorbent and enzymatic amplification) Either antigen or antibody is bound to a solid substrate (polystyrene surface), and an enzyme-conjugated antibody is added, the a substrate ELISA may be run in a qualitative or quantitative format is biochemical technique used mainly in immunology can be either qualitative or quantitative In ELISA, there are three necessary reagents: i. antibody coupled to solid supports, ”immunosorbent” ii, antigen or antibody which is marked with enzyme and is called conjugate if, substrate ELISA combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme ELISA can be used to detect the presence of antigens that are recognized by an antibody or it can be used to test for antibodies that recognize an antigen 35; | Principle of ELISA 1. The basic principle of an ELISA is to use an ~_ 2. enzyme to detect the binding of antigen Macatee (Ag) antibody (Ab) n + Abor Ag could be Antibody conjugated/ complexed with an 1d antibody-enzyme conjugate enzyme such as alkaline phosphatase, horse radish peroxidase etc Eyryme 2. The enzyme convertsa colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding + Color intensity is measured spectrophotometrically (optical Substrate densities, OD) Ada substrate, © (eolertess) 3, An ELISA can be used to detect either the presence of Ags or Abs in a sample, depending on how the testis designed. 2, 4. Various designs exist such as vo simple/direct, indirect, sandwich and competitive Types of ELISA Indirect Apply a sample of known antigen of known concentration to a microtiter plat. Block non-specific adsorption of other proteins to the plate using non-interacting protein e.g. BSA, casein, serum. Coat the plate with serum sample of unknown antigen concentration diluted ina be, ‘Ada detection antibody specific to the antigen of interest and wash off unbound antibodies. ‘+ Introduce a 2° antibody specific to the detection antibody. The 2° antibody is usually conjugated to the substrate-specific enzyme, Omit this step if the detection antibody is conjugated. Wash the plate and apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal. Quantify using a spectrophotometer, spectrofluorometer, or other optical electrochemical device. Major disadvantage: antigen immobilization is non-specific 362. Sandwich Prepare a surface to which a known quantity of capture antibody is bound. Block any non specific binding sites on the surface Apply the antigen-containing sample to the plate Wash the plate, so that unbound antigen is removed Apply primary antibodies that bind specifically to the antigen Apply enzyme-linked secondary antibodies which are specific to the primary antibodies Wash the plate, so that the unbound antibody-enzyme conjugates are removed Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal Measure the absorbency or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen. 3. Competitive Unlabeled antibody is incubated in the presence of its antigen These bound antibody /antigen complexes are then added to an antigen coated well The plate is washed, so that unbound antibody is removed. (the more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.") ‘The 2° antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal © NB: Less is more (more antigen in your sample means more Ab competed away) 4, Reverse (Reading Assignment: read and make short notes on the layout of reverse elisa and provide its advantages over the other types) Advantages of ELISA i. specificity i. quantitativ Applications of ELISA i ii, iv. Serum Antibody Concentrations Detecting potential food allergens (milk, peanuts, walnuts, almonds and eggs) Disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common, colds, cholera, STD ete Detections of antigens e.g. pregnancy hormones, drug allergen, GMO, mad cow disease 37v. Detection of antibodies in blood sample for past exposure to disease e.g. Lyme Disease, trichinosis, HIV, bird flu 38PROTEIN CHARACTERIZATION METHODS. ‘Western Blot on Proteins © aprocedure for using antibodies to detect proteins after separation by PAGE © Western blotting is a very good method to prove that the protein found in an SDS-PAGE gel is the protein you are interested in, if a specific antibody is available to use for detecting your protein © the "blot" is prepared as a replica of the SDS-PAGE gel since the antibody detection process cannot be directly carried out on the gel itself © the blot is prepared using a protein binding membrane e.g nitrocellulose, PVDF et. © to prepare a Western Blot, small amounts of protein of interest are denatured by the usual procedure using SDS and heating in the presence of 2- mercaptoethanol, © the denatured protein samples along with standard protein molecular weight markers which have been dyed to aid in detection on the blot, are electrophoresed as usual in an SDS-PAGE gel. © then the gel is placed on a piece of nitrocellulose membrane and the proteins are electrophoretically transferred from the gel to the nitrocellulose membrane, © next the membrane is blocked with a protein solution (like BSA or fat-free milk) to prevent non-specific binding of the antibody to the membrane. © next the membrane blot is incubated in the specific antibody (primary antibody) for the protein of interest. © this is followed by a binding step where the antibody is allowed to bind another antibody (secondary antibody) with specificity against the first (for 39example if the primary antibody came from a rabbit, then the secondary antibody would be goat antibody prepared against rabbit immunoglobulin). © the secondary antibody is conjugated to an enzyme which will make it possible to detect it © after the secondary antibody is bound and excess washed away, the substrate for the conjugated enzyme are added and the color reaction developed on the blot. co Western blotting is a very sensitive (as little as 1 nanogram of protein can be detected on the blot). © since the primary antibody used is specific for the protein of interest, the colored bands found on the blot represent evidence for the presence of the specific protein of interest, Microarray technology (protein detection method) microarray is a relatively new technology that involves analytical method which, compares a person's DNA to “control DNA” also called Gene chips analysis in which thousands of DNA pieces in genes are spotted on a small surface enabling examination of changes in gene expression profile of thousands of genes in one experiment GeneChip is a brand of Affymetrix like a Southern or northem blot on steroids © instead of a single probe, >1,000,000 probes a tiny 1 em? piece of glass is divided into a grid of as many as 1,000,000 spots or features each microarray is manufactured with a specific set of features/ probes for a defined application on each spot, a 25 nt ssDNA sequence is attached / built up "for each spot, itis a unique sequence but present in millions of copies on that spot the sample to be tested must be labeled so cDNA or cRNA is made and a label incorporated + often labeled with biotin 40+ the labeled sample is added to the array and allowed to hybridize to whichever features it is complementary to ‘+ unbound sample is washed away ‘+a computer detects on which features/spots the sample (DNA) has hybridized ‘signal intensity is measured: Quantitative analysis ‘+ sensitive enough to detect single base differences between probe and sample Applications: gene expression studies (look at expression of ALL genes ina genome, including alternatively spliced variants) * genotyping (to detect presence of an organism's or individual's DNA) a1Protein Structure and Function + determining the amino acid sequence of a protein used to be a very laborious time consuming process involving chemical and enzymatic degradation. + today, the amino acid sequence of proteins is usually determined from the nucleotide sequence of the gene, a relatively simple and rapid process. + the AA sequence of the same protein from many sources, e.g., cytochrome ¢, shows that some amino acid residues are conserved among all the proteins, whereas others vary. ‘+ such a sequence comparison provides valuable information about possible roles of specific amino acid residues that may be either + essential for protein's function, or + essential for protein's structure (e.g,, residues needed for correct tertiary folding, or needed for interacting with another subunit + tertiary structure decides the biochemical function of a protein (most active form) + change in 3-D structure usually results in a loss of function. + knowing the 3-D structure of a protein is the key to understanding its function and to protein engineering. ‘+ finding the primary structure is easy + finding the secondary structure is difficult, + finding the tertiary structure is very difficult + finding the function of a protein is difficult Primary structure = sequence of AAs Secondary structure = folding of parts of the AA chain + Tertiary structure = real 3-D conformation (Quaternary Structure = 3-D arrangement of several domains Primary structure determination + Step 1: Amino Acid Analysis Protein is hydrolyzed in strong acid or base at high temperatures Mixture of amino acids generated by protein hydrolysis is analyzed by chromatography + Step 2: Sequence Analysis, protein cleaved into polypeptides polypeptides subjected to amino acid analysis or Edman degradation a2+ Edman Degradation: © One residue at a time from the amino terminus can be chemically derivatized, removed and identified. © The antino-terminal amino acid residue is derivatized and removed (cleaved off) for subsequent identification, leaving the peptide or protein one resistue shorter, for another round of derivatization, removal and identification of the next amino acid residue in the sequence, etc., for up to about 75 residues from the amino terminus) © Coupling (labeling): chemical modification of the | -amino group of a peptide or protein by the Edman reagent (PITC, phenylisothiocyanate) + Cleavage ("release"): anfydrous acid (c.g., anhyrous trifluroacetic acid) + Conversion of the initial unstable derivative to the more stable phenylthiohydantoin derivative for identification (by chromatographic behavior and comparison with known. PTH standards) + Whole procedure has been automated, done by a single machine (sequenator), with output to a computer + Whatis needed are smaller fragments, with new amino termini, which can be individually purified and sequenced. ‘+ This is accomplished by cleaving the protein with a proteolytic enzyme, such as trypsin, or a chemical reagent such as cyanogen bromide, which generates a set of peptides, fragments of the original protein, that can be separated and sequenced Sequence analysis ‘+ ASpecific Chemical Cleavage: © Cyanogen bromide (Br-CN): cleaves peptide bonds formed by carboxyl of ‘methionine + Specific Enzymatic Cleavages: © Trypsin: cleaves peptide bonds formed by carboxyl of lysine and arginine © Chymotrypsin: cleaves peptide bonds formed by carboxyl of phenylalanine, tyrosine, and tryptophan ‘+ Sequential Degradation: © Edman Degradation: cleaves n-terminal aa shortened pede HO . das max 2 ay yy OF GMCS py mare o ojJH#oa oN HOO hd SXcais ond : a phenylthiokydantoin 43‘valine glycine alanine-methionine-leusine-profine 49, Xray Diffraction + used to determine the arrangement of atoms in a solid compounds and to measure bond lengths and structures. + Diffraction: interference between scattered waves, which arises when there is an object in their path. Note: For diffraction to be observed, the wavelength of radiation must be about equal to the distances between the atoms. X-rays are used because they have short wavelengths, which correspond to the bond length. + Acollection of atoms produces complex interference depending upon bond lengths and angles.) Procedure and Instrumentation: i, Grow Crystal: a challenging process. ‘+ must be perfect - no twinning, or inclusions; small (0.1-0.5mm) ii, Place crystal in four-circle diffractometer: a device for rotating the crystal and the detector so that the entire diffraction pattern can be recorded under the control of a computer. + X-rays are beamed at the crystals, electrons diffract the x-rays, producing, diffraction patterns, + because of the crystals’ highly ordered and repetitive structure, many X- rays © diffracting off many electron clouds in approximately the same relative position and orientation throughout the crystal will result in constructive interference and give a detectable signal Fourier synthesis is used to analyze the intensities of the x-rays at all the settings of the angles in the diffractometer, and convert these measurements into the locations of the atoms. iv. From these informations, the Electron Density Map is created. It shows the contour lines of electron density, and therefore, provides location of atoms relative to each other. 44Smaller circle = higher electron density Center of circles = atom Bond angles and bond lengths may be determined to get the position of atoms in space. Hydrogen atoms often cannot be located because they contain only 1 electron and Nuclear Magnetic Resonance (NMR) Spectroscopy + Basis: © aspinning charged particle (a nucleus) behaves as a magnet, and can interact with an externally imposed magnetic field such that absorbance of electromagnetic radiation of appropriate energy (in the microwave, ie. radio, frequency range) can flip the spin. © nuclei used in biochemical studies include 'H (proton NMR), 2H, 8C, N, 70, &P, and ¥F (in "E-Tyr). © toget an NMR spectrum (in ppm), you “sit” on a magnetic field strength and change the radio frequency to get resonance. ‘The type of nucleus you're observing, but also the molecular structural environment of the nucleus (including its solvent and surroundings in 3 dimensional space) affect the width and position (position = "chemical shift") of the NMR signal (peak) for that nucleus, Interaction with a nearby nucleus within the molecule can cause spin coupling, which is seen as splitting of the NMR signal (double peak). Altering the spin on one nucleus can affect the spin on a nearby nucleus (<~5A away), and for small proteins it is possible by NMR to do enough distance measurements between nuclei within the tertiary structure to determine the entire 3-climensional structure. © Uses of NMR complete 3-D structure of small proteins in solution (<25,000 daltons) conformational changes (e-g., during folding) determination of pK, of an ionizable group, e.g. His, follow ligand binding dynamics (motion in solution), e.g. Tyr and Phe ring flips 45who controls the past controls the future, who controls the present controls the past. By Robert T. Kiyosaki V4.0(9-9-2002) If you find and correct errors in the text, please update the version number by 0.1 and. redistribute. Ripped by Tangtang INTRODUCTION There is a Need Does school prepare children for the real world? "Study hard and get good grades and you will find a high-paying job with great benefits," my parents used to say. Their goal in life was to provide a college education for my older sister and me, so that we would have the greatest chance for success in life When T finally earned my diploma in 1976-graduating with honors, and near the top of my class, in accounting from Florida State University-my parents had realized their goal. It was the crowning achievement of their lives. In accordance with the "Master Plan," I was hired by a “Big 8" accounting firm, and I looked forward to a long reer and retirement at an early age. My husband, Michael, followed a similar path. We both came from hard- working families, of modest means but with strong work ethics. Michael also graduated with honors, but he did it twice: £ ¢ as an engineer and then from law school. He was quickly recruited by a prestigious Washington, D.C., law firm that specialized in patent law, and his future seemed bright, career path well- defined and early retirement guaranteed. Although we have been successful in our careers, they have not turned out quite as we expected. we both have changed positions several times-for all the right reasons-but there are no pension plans vesting on our behalf. Our retirement funds are growing only through our individual contributions. Michael and T have a wonderful marriage with three great children. As T write this, two are in college and one is just beginning high school. we havewho controls the past controls the future, who controls the present controls the past. spent a fortune making sure our children have received the best education available. one day in 1996, one of my children came home disillusioned with school. He was bored and tired of studying. "why should 1 put time into studying subjects I will never use in real life?" he protested. Without thinking, T responded, "Because if you don't get good grades, you won't get into college." Regardless of whether I go to college," he replied, "I'm going to be rich." "IE you don't graduate from college, you won't get a good job," I responded with a tinge of panic and motherly concern. "And if you don't have a good job, how do you plan to get rich?" My son smirked and slowly shook his head with mild boredom. We have had this talk many times before. He lowered his head and rolled his eyes. My words of motherly wisdom were falling on deaf ears once again. Though smart and strong-willed, he has always been a polite and respectful young man Mom," he began. Tt was my turn to be lectured. “Get with the times! Look around; the richest people didn't get rich because of their educations. Look at Michael Jordan and Madonna. Even Bill Gates, who dropped out of Harvard, founded Microsoft; he is now the richest man in America, and he's still in his 30s. There is a baseball pitcher who makes more than $4 million a year even though he has been labeled “mentally challenged.' " There was a long silence between us. Tt was dawning on me that T was giving my son the same advice my parents had given me. The world around us has changed, but the advice hasn't Getting a good education and making good grades no longer ensures success, and nobody seems to have noticed, except our children. "Mom," he continued, "I don't want to work as hard as you and dad do. You make a lot of money, and we live in a huge house with lots of toys. If I follow your advice, I'll wind up like you, working harder and harder only to pay more taxes and wind up in debt. There is no job security anymore; I know all about downsizing and rightsizing. I also know that college graduates today earn less than you did when you graduated. Look at doctors. They don't make nearly as much money as they used to. I know I can't rely on Social Security or company pensions for retirement. I need new answers. He was right. He needed new answers, and so did I. My parents’ advice may have worked for people born before 1945, but it may be disastrous for those ofwho controls the past controls the future, who controls the present controls the past. us born into a rapidly changing world. No longer can I simply say to my children, "Go to school, get good grades, and look for a safe, secure job." I knew I had to look for new ways to guide my children's education As a mother as well as an accountant, I have been concerned by the lack of financial education our children receive in school. many of today's youth have credit cards before they leave high school, yet they have never had a course in money or how to invest it, let alone understand how compound interest works on credit cards. Simply put, without financial literacy and the knowledge of how money works, they are not prepared to face the world that awaits them, a world in which spending is emphasized over savings. When my oldest son became hopelessly in debt with his credit cards as a freshman in college, I not only helped him destroy the credit cards, but I also went in search of a program that would help me educate my children on financial matters. one day last year, my husband called me from his office. "I have someone I think you should meet," he said. "His name is Robert Kiyosaki. He's a businessman and investor, and he is here applying for a patent on an educational product. I think it's what you have been looking for." Just what I Was Looking For My husband, Mike, was so impressed with CASHFLOW, the new educational product that Robert Kiyosaki was developing, that he arranged for both of us to participate in a test of the prototype. Because it was an educational game, T also asked my 19-year-old daughter, who was a freshman at a local university, if she would like to take part, and she agreed. About fifteen people, broken into three groups, participated in the test. Mike was right. It was the educational product T had been looking for. But it had a twist: Tt looked like a colorful monopoly board with a giant well- dressed rat in the middle. Unlike Monopoly, however, there were two tracks: inside and one outside. The object of the game was to get out of the inside track-what Robert called the "Rat Race" and reach the outer track, or the "Fast Track." As Robert put it, the Fast Track simulates how rich people play in real life. Robert then defined the "Rat Race" for us "IE you look at the life of the average-educated, hard-working person, there is a similar path. The child is born and goes to school. The proud parents are excited because the child excels, gets fair to good grades, and is acceptedwho controls the past controls the future, who controls the present controls the past. into a college. The child graduates, maybe goes on to graduate school and then does exactly as programmed: looks for a safe, secure job or career. The child finds that job, maybe as a doctor or a lawyer, or joins the Army or works for the government. Generally, the child begins to make money, credit cards start to arvive in mass, and the shopping begins, if it alzeady hasn't “Having money to burn, the child goes to places where other young people just like them hang out, and they meet people, they date, and sometimes they get married. Life is wonderful now, because today, both men and women work. Two incomes are bliss. They feel successful, their future is bright, and they decide to buy a house, a car, a television, take vacations and have children. The happy bundle arrives. The demand for cash is enormous. The happy couple decides that their careers are vitally important and begin to work harder, seeking promotions and raises. The raises come, and so does another child and the need for a bigger house. They work harder, become better employees, even more dedicated. They go back to achool to get more specialized skills so they can earn more money. Maybe they take a second job. Their incomes go up, but so does the tax bracket they're in and the real estate taxes on their new large home, and their social security taxes, and all the other taxes. They get their large paycheck and wonder where all the money went. They buy some mutual funds and buy groceries with their credit card. The children reach 5 or 6 years of age, and the need to save for college increases as well as the need to save for their retirement. “That happy couple, born 35 years ago, is now trapped in the Rat Race for the rest of their working days. They work for the owners of their company, for the government paying taxes, and for the bank paying off a mortgage and credit cards. then, they advise their own children to “study hard, get good grades, and find a safe job or career.’ They learn nothing about money, except from those who profit from their naiveté, and work hard all their lives. The process repeats into another hard-working generation. This is the “Rat Race'." The only way to get out of the "Rat Race" is to prove your proficiency at both accounting and investing, arguably two of the most difficult subjects to master. As a trained CPA who once worked for a Big 8 accounting firm, I was surprised that Robert had made the learning of these two subjects both fun and exciting. The process was so well disguised that while we were diligently working to get out of the "Rat Race," we quickly forgot we were learning. Soon a product test turned into a fun afternoon with my daughter, talking about things we had never discussed before. as an accountant, playing a game that required an Income Statement and Balance Sheet was easy. So I had the timewho controls the past controls the future, who controls the present controls the past. to help my daughter and the other players at my table with concepts they did not understand. I was the first person-and the only person in the entire test group- to get out of the “Rat Race” that day. I was out within 50 minutes, although the game went on for nearly three hours At my table was a banker, a business owner and a computer progranmer. what greatly disturbed me was how little these people knew about eit her accounting or investing, subjects so important in their lives. 1 wondered how they managed their own financial affairs in real life. 1 could understand why my 19-year-old daughter would not understand, but these were grown adults, at least twice her age. After I was out of the “Rat Race," for the next two hours T watched my daughter and these educated, affluent adults roll the dice and move their markers. Although I was glad they were all learning so much, I was disturbed by how much the adults did not know about the basics of simple accounting and investing. They had difficulty grasping the relationship between their Income Statement and their Balance Sheet. As they bought and sold assets, they had trouble remembering that each transaction could impact their monthly cash flow I thought, how many millions of people are out there in the real world struggling financially, only because they have never been taught these subjects? Thank goodness they're having fun and are distracted by the desire to win the game, I said to myself. After Robert ended the contest, he allowed us fifteen minutes to discuss and critique CASHFLOW among ourselves. The business owner at my table was not happy. He did not like the game. "I don't need to know this," he said out loud. "I hire accountants, bankers and attorneys to tell me about this stuff." To which Robert replied, "Have you ever noticed that there are a lot of accountants who aren't rich? And bankers, and attorneys, and stockbrokers and real estate brokers. They know a lot, and for the most part are smart people, but most of them are not rich. Since our schools do not teach people what the rich know, we take advice from these people. But one day, you're driving down the highway, stuck in traffic, struggling to get to work, and you look over to your right and you see your accountant stuck in the same traffic jam. You look to your left and you see your banker. That should tell you something. The computer progranmer was also unimpressed by the game: "I can buy software to teach me this.” The banker, however, was moved. "I studied this in school-the accounting part, that is-but I never knew how to apply it to real life. Now I know. T need to get myself out of the “Rat Race." ™who controls the past controls the future, who controls the present controls the past. But it was my daughter's comments that most touched me. "I had fun learning," she said. "I learned a lot about how money really works and how to invest." Then she added: "Now I know I can choose a profession for the work I want to perform and not because of job security, benefits or howmuch I get paid. If T learn what this game teaches, I'm free to do and study what my heart wants to study. . .rather than study something because businesses are looking for certain job skills. If 1 learn this, I won't have to worry about job security and social Security the way most of my classmates already do I was not able to stay and talk with Robert after we had played the game, but we agreed to meet later to further discuss his project. T knew he wanted to vse the game to help others become more financially savvy, and I was eager to hear more about his plans. My husband and I set up a dinner meeting with Robert and his wife within the next week. Although it was our first social get-together, we felt as if we had known each other for years. We found out we had a lot in common. We covered the gamut, from sports and plays to restaurants and socio-economic issues. We talked about the changing world. We spent a lot of time discussing how most Americans have little or nothing saved for retirement, as well as the almost bankrupt state of Social Security and Medicare. Would my children be required to pay for the retirement of 75 million baby boomers? We wondered if people realize how risky it is to depend on a pension plan. Robert's primary concern was the growing gap between the haves and have nots, in America and around the world. A self-taught, self-made entrepreneur who traveled the world putting investments together, Robert was able to retire at the age of 47. He came out of retirement because he shares the same concern T have for my own children. He knows that the world has changed, but education has not changed with it. According to Robert, children spend years in an antiquated educational system, studying subjects they will never use, preparing for a world that no longer exists. "Today, the most dangerous advice you can give a child is “Go to school, get good grades and look for a safe secure job,' " he likes to say. "That is old advice, and it's bad advice. If you could see what is happening in Asia, Europe, South America, you would be as concerned as I am."who controls the past controls the future, who controls the present controls the past. It's bad advice, he believes, “because if you want your child to have a financially secure future, they can't play by the old set of rules. It's just too risky." T asked him what he meant by "old rules?" people like me play by a different set of rules from what you play by, he said. “What happens when a corporation announces a downsizing?" “People get laid off,” I said. "Families are hurt. Unemployment goes up." Yes, but what happens to the company, in particular a public company on the stock exchange? That's right," he said. “shareholders, get richer. ‘That is what I mean by a different set of rules. Employees lose; owners and investors win." Robert was describing not only the difference between an employee and employer, but also the difference between controlling your own destiny and giving up that control to someone else. put it's hard for most people to understand why that happens," I said "They just think it's not fair ‘That's why it is foolish to simply say to a child, “Get a good education,' "he said. "It is foolish to assume that the education the school system provides will prepare your children for the world they will face upon graduation. Each child needs more education. Different education. And they need to know the rules. The different sets of rules. “There are rules of money that the rich play by, and there are the rules that the other 95 percent of the population plays by," he said. "And the 95 percent learns those rules at home and in school. That is why it's risky today to simply say to a child, “Study hard and look for a job.' A child today needs a more sophisticated education, and the current system is not delivering the goods. T don't care how many computers they put in the classroom or how much money oon So how does a parent teach their children, what the school does not? How do you teach accounting to a child? Won't they get bored? And how do you teach investing when as a parent you yourself are risk averse? Instead of teaching mywho controls the past controls the future, who controls the present controls the past. children to simply play it safe, I decided it was best to teach them to play it omart. "So how would you teach a child about money and all the things we've talked about?" I asked Robert. "How can we make it easy for parents especially when they don't understand it themselves?" "I wrote a book on the subject, " he said. “Where is it?" "In my computer. It's been there for years in random pieces. I add to it occasionally but I've never gotten around to put it all together. I began writing it after my other book became a best seller, but I never finished the new one. It's in pieces." And in pieces it was. After reading the scattered sections, I decided the book had merit and needed to be shared, especially in these chant ng times. We agreed to co-author Robert's book. I asked him how much financial information he thought a child needed. He said it would depend on the child. He knew at a young age that he wanted to be rich and was fortunate enough to have a father figure who was rich and willing to guide him. Education is the foundation of success, Robert said. Just as scholastic skills are vitally important, so are financial skills and communication skills. What follows is the story of Robert's two dads, a rich one and a poor one, that expounds on the skills he's developed over a lifetime. The contrast between two dads provides an important perspective. The book is supported, edited and assembled by me. For any accountants who read this book, suspend your academic book knowledge and open your mind to the theories Robert presents. Although many of them challenge the very fundamentals of generally accepted accounting principles, they provide a valuable insight into the way true investors analyze their investment decisions. when we as parents advise our children to "go to school, study hard and “get a good job," we often do that out of cultural habit. It has always been the right thing to do. When I met Robert, his ideas initially startled me. Raving been raised by two fathers, he had been taught to strive for two different goals. His educated dad advised him to work for a corporation. His rich dad advised him to own the corporation. Both life paths required education, but the subjects of study were completely different. His educated dad encouraged Robert to be a smart person. His rich dad encouraged Robert to know how to hire smart people. Having two dads caused many problems. Robert's real dad was the superintendent of education for the state of Hawaii. By the time Robert was 16,who controls the past controls the future, who controls the present controls the past. education would also be a benefit to hii Truthfully, the ideas in this book are probably too far fetched and radical for most parents today. Some parents are having a hard enough time simply keeping their children in school. But in light of our changing times, as parents we need to be open to new and bold ideas. To encourage children to be employees is to advise your children to pay more than their fair share of taxes over a lifetime, with little or no promise of a pension. And it is true that taxes are a person's greatest expense. New ideas are needed and this book provides them Robert claims that the rich teach their children differently. They teach their children at home, around the dinner table. These ideas may notbe the ideas you choose to discuss with your children, but thank you for looking at them. And T advise you to keep searching. In my opinion, as a mom and a CPA, the concept of simply getting good grades and finding a good job is an old idea. We need to advise our children with a greater degree of sophistication. We need new ideas and different education. Maybe telling our children to strive to be good employees while also striving to own their own investment corporation is not such a bad idea It is my hope as a mother that this book helps other parents. Tt is Robert's hope to inform people that anyone can achieve prosperity if they so choose. If today you are a gardener or a janitor or even unemployed, you have the ability to educate yourself and teach those you love to take care of themselves financially. Remember that financial intelligence is the mental process via which we solve our financial problems. Today we are facing global and technological changes as great or even greater than those ever faced before. No one has a crystal ball, but one thing is for certain: Changes lie ahead that are beyond our reality. Who knows what the future brings? But whatever happens, we have two fundamental choices: play it safe or play it smart by preparing, getting educated and awakening your own and your children's financial genius. - Sbaron Lechterwho controls the past controls the future, who controls the present controls the past. For a FREE AUDIO REPORT "What My Rich Dad Taught Me About Money" all you have to do is visit our special website at www.richdadbooki.com and the report is yours free Thank you Rich Dad, Poor Dad 1. CHAPTER ONE Rich Dad, Poor Dad As narrated by Robert Kiyosaki As a young boy, having two strong fathers both influencing me was difficult. I wanted to be a good son and listen, but the two fathers did not saywho controls the past controls the future, who controls the present controls the past. the same things. The contrast in their points of view, particularly where money was concerned, was so extreme that I grew curious and intrigued. I began to start thinking for long periods of time about what each was saying Much of my private time was spent reflecting, asking myself questions such as, "Why does he say that?" and then asking the same question of the other dad's statement. Tt would have been much easier to simply say, "Yeah, he's right. T agree with that." Or to simply reject the point of view by saying, “The old man doesn't know what he's talking about." Instead, having two dads whom I loved forced me to think and ultimately choose a way of thinking for myself. As a process, choosing for myself turned out to be much more valuable in the long run, rather than simply accepting or rejecting a single point of view. One of the reasons the rich get richer, the poor get poorer, and the middle class struggles in debt is because the subject of money is taught at home, not in school. Most of us learn about money from our parents. So what can a poor parent tell their child about money? They simply say "Stay in school and study hard." The child may graduate with excellent grades but with a poor person's financial programming and mind-set. Tt was learned while the child was young, Money is not taught in schools. Schools focus on scholastic and professional skills, but not on financial skills. This explains how smart bankers, doctors and accountants who earned excellent grades in school may still struggle financially all of their lives. Our staggering national debt is due in large part to highly educated politicians and government officials making financial decisions with little or no training on the subject of money. I often lock ahead to the new millennium and wonder what will happen when we have millions of people who will need financial and medical assistance. They will be dependent on their families or the government for financial support. What will happen when Medicare and Social Security run out of money? How will a nation survive if teaching children about money continues to be left to parents- most of whom will be, ox already are, poor? Because I had two influential fathers, I learned from both of them. I had to think about each dad's advice, and in doing so, T gained valuable insight into the power and effect of one's thoughts on one's life. For example, one dad had a habit of saying, The other dad a statement, and the other is a question. One lets you off the hook, and the other forces you to think. My soon-to-be-rich dad would explain that by automatically saying the words "I can't afford it," your brain stops working. By asking the question "How can I afford it?" your brain is put to work. He did notwho controls the past controls the future, who controls the present controls the past. mean buy everything you wanted. He was fanatical about exercising your mind, the most powerful computer in the world. "My brain gets stronger every day because I exercise it. The stronger it gets, the more money I can make." He believed that automatically saying "I can't afford it" was a sign of mental laziness Although both dads worked hard, I noticed that one dad had a habit of putting his brain to sleep when it came to money matters, and the other had a habit of exercising his brain. The long-term result was that one dad grew stronger financially and the other grew weaker. rt is not much different from a person who goes to the gym to exercise on a regular basis versus someone who sits on the couch watching television. Proper physical exercise increases your chances for health, and proper mental exercise increases your chances for wealth. Laziness decreases both health and wealth. My two dads had opposing attitudes in thought. One dad ‘The other ‘One dad recommended, The other recommended, " one dad said, other said, one encouraged talking about money and business at the dinner ,table. The other forbade the subject of money to be discussed over a meal. one said, The one believed, The other believed, Both dads paid their bills on time, yet one paid his bills first while the other paid his bills last. one dad believed in a company or the government taking care of you and your needs. He was always concerned about pay raises, retirement plans, medical benefits, sick leave, vacation days and other perks. He was impressed with two of his uncles who joined the military and earned a retirement and entitlement package for life after twenty years of active service. He loved the idea of medical benefits and PX privileges the military provided its retirees. He also loved the tenure system available through the university. The idea of job protection for life and job benefits seemed more important, at tines, than the job. He would often say, "I've worked hard for the government, and I'm entitled to these benefits.”who controls the past controls the future, who controls the present controls the past. The other believed in total financial self-reliance. He spoke out against the “entitlement” mentality and how it was creating weak and financially needy people. He was emphatic about being financially competent one dad struggled to save a few dollars. The other simply created investments. one dad taught me how to write an impressive resume so I could find a good job. The other taught me how to write strong business and financial plans so T could cxeate jobs. Being a product of two strong dads allowed me the luxury of observing the effects different thoughts have on one's life. I noticed that people really do shape their life through their thoughts (BEOBREBY|BeCamE)ZEBIGEY, My rich dad, on the other hand, always referred to himself as rich. He would say things like, (Fiim|al/=ieH|many |and|icH|peSpIS) don't do this.” Even when he was flat broke after a major financial setback, he continued to refer to himself as a rich man. He would cover himself by saying, "There is a difference between being poor and being broke. - Broke is temporary, and poor is eternal.” My poor dad would also say, “I'm not interested in money," or "Money doesn't matter." My rich dad always said, "Money is power The power of our thoughts may never be measured or appreciated, but it became obvious to me as a young boy to be aware of my thoughts and how T expressed myself. T noticed that my poor dad was poor not because of the amount of money he earned, which was significant, but because of his thoughts and actions. As a young boy, having two fathers, T became acutely aware of being careful which thoughts I chose to adopt as my own. Whom should I listen to-my rich dad or my poor dad? disagreed in what they thought was important to learn. one wanted me to study one with all the college degrees.who controls the past controls the future, who controls the present controls the past. A Lesson From Robert Frost Robert Frost is my favourite poet. Although I love many of his poems, my favorite is The Road Not Taken. I use its lesson almost daily: The Road Not Taken vo roads diverged in a yellow wood, And sony £ could not teavol both and be one teaveles, long I stood And locked down one as far a8 I could Zo whose it bent in the undergrowth: then took the other, oa just as fair, And having perhaps the better clain, Beceuse Jt was gressy and wented wear Though as for thet the passing there Had worn then really about the sane, aod both that notning equally lay In leaves no step nad troasen black. Oh, x kept the first for another day! Yet knowing how way leads onto way, X doubted Af 5 should ever cone back Z shall be telling this with a sigh Sonewhere ages and ages hence: Ho roads diverged in a wood, id] B60k) the! ie liess|EraveledEy and chet ail the Robert Frost (1316) ifference. And that made all the difference. over the years, I have often reflected upon Robert Frost's poem. Choosing not to listen to my highly educated dad's advice and attitude about money was a painful decision, but it was 2 decision that shaped the rest of my life once I made up my mind whom to listen to, my education about money began. My rich dad taught me over a period of 30 years, until I was age 39. He stopped once he realized that 1 knew and fully understood what he had been trying to (GBisaELom, Noney cones and goes, but if you have the education aboot how money Works, you gain power over it and can beyin building wlth, The essen positive drum into my often thick skull. Money is one form of power thinking alone does not work is because most people went to school and never learned how money works, so they spend their lives working for money. Because I was only 9 years old when I started, the lessons my rich dad taught me were simple. And when it wes all said and done, (See S=SISRiieSi® (GBURIHERRERB, epeaced over 30 years. This book {9 about those six lessons, put as simply as possible as my rich dad put forth those lessons to me. The lessonswho controls the past controls the future, who controls the present controls the past. are not meant to be answers but guideposts. Guideposts that will assist you and your children to grow wealthier no matter what happens in a world of increasing change and uncertainty. Lesson #1 The Rich Don't Work for Money Lesson #2 Why Teach Financial Literacy? Lesson #3 Mind Your own Business Lesson #4 The History of Taxes and the Power of Corporations Lesson #5 The Rich Invent Money Lesson #6 Work to Learn Don't Work for Money 2. CHAPTER TWO Lesson One: The Rich Don't Work For Money “Dad, Can You Tell Me How to Get Rich?" My dad put down the evening paper. “why do you want to get rich, son?" "Because today Jimny's mom drove up in their new Cadillac, and they were going to their beach house for the weekend. He took three of his friends, but Mike and I weren't invited. They told us we weren't invited because we were ‘poor kids!" wThey did?" my dad asked incredulously. "Yeah, they did." I replied in a hurt tone. My dad silently shook his head, pushed his glasses up the bridge of his nose and went back to reading the paper. I stood waiting for an answer. The year was 1956. T was 9 years old. By some twist of fate, T attended the same public school where the rich people sent their kids. We were primarily a sugar plantation town. The managers of the plantation and the other affluent people of the town, such as doctors, business owners, and bankers, sent their children to this school, grades 1 to 6. After grade 6, their children were generally sent off to private schools. Because my family lived on one side of the street, I went to this school. Had I lived on the other side of the street, T would have gone to a different school, with kids from families more like mine After grade 6,these kids and I would go on to the public intermediate and high school. There was no private school for them or for me. My dad finally put down the paper. I could tell he was thinking. well, son," he began slowly. "If you want to be rich, you have to learn to make money.”