ELECTROPHORESIS

Done By: Samyah Alanazi

lecture Outline
• 1- Introduction.

• 2- Principle.
• 3- Factors affecting the rate of migration . • 4- solutions. • 5- Principal of electro-endosmosis. • 6- Factors affecting electrophoresis mobility. • 7- Instrumentation. • 8-Types of electrophoresis.

Introduction
• The term electrophoresis describes the migration of a

charged particle under the influence of an electric field.
• Many important biological molecules, such as amino acids

,peptides ,proteins ,nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations (+) or anoins (-).
• Under the influence of an electric field these charged

particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.

: E*q V = --------------f . properties of the support medium. size. pH . shape . time frame of the procedure. temp. This can be represented by following eq.Principle Any charged ion or molecule migrates when placed in an electric field. The rate of migration depend upon its net charge. of the operating system and the applied electric current.

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E = electric field in volts per cm q = net electric charge on the molecule f = frictional coefficient The movement of charged particle in an electric field is expressed in terms of electrophoretic mobility . it varies with size. . Thus electrophoretic mobility (µ) of a molecule is directly proportional to charge density(charge\mass ratio).denoted by µ. f doesn’t vary however. µ = q/f For molecules with similar shapes.• • • • v = velocity of migration of the molecule. where.

Thus. a small proportion are being conducted by the sample ions.• The electric field is removed before the molecules in the sample reach the electrodes. • The current in the solution between the electrodes is conducted mainly by the buffer ions.voltage (V) and resistance ( R ): • R = V/I. The sample then are then located by staining with an appropriate dye. Ohm’s law express the relationship between current (I). the components will have been separated according to their electrophoretic mobility. electrophoresis is an incomplete form of electrolysis . .

The distance migrated by the ions will be proportional to both current and time. .• It is therefore appears that it is possible to accelerate an electrophoretic separation by increasing the applied voltage. which would result in a corresponding increase the electric flowing .

2. larger particles have smaller electrophoretic mobility compared to smaller particles.FACTORS AFFECTING MIGRATION RATE 1. 3. It depends on buffer pH and PI.Net Charge – higher the charge greater the electrophoretic mobility. Therefore globular protein move faster than fibrous protein.Size – bigger the molecule greater are the frictional and electrostatic forces exerted on it by the medium.Shape – rounded contours elicit lesser frictional and electrostatic retardation compared to sharp contours. . Consequently.

• 6. increase viscosity will slower the movement.time.Temp. • 5.frame of the procedure: • longer time: Sample would've ran off the gel.• 4. being merged and unable to be read • 7.Buffer pH: it has been chosen in order to maintain net charge of a molecule/atom.prosperities of support medium: • A higher pore size will increase mobility while. of the system: • Increase heat lead to the followings: . • shorter time: not far enough and would have caused the bands to not separate enough.

which leads to mixing of separated samples. This may include denaturation of proteins (and thus the lose of enzyme activity). and hence a reduction in the resistance of the medium. . • D.the formation of convection currents. • B.A decrease of buffer viscosity. • C-Thermal instability of samples that are rather sensitive to heat.Heat effects on the particle mobility • A.An increased rate of diffusion of sample and buffer ions leading to broadening of separated sample.

• 8- final factor affecting separation is called electroendosmosis which is due to presence of charged groups on the surface of support medium . .solutions • 1.Using a stabilised power supply which provides constant power and thus eliminates fluctuations in heating .

These groups become ionized in basic and neutral buffers: in the electric field they will be attracted by the anode.g. the gel) and/or the surface of the separation equipment such as glass plates. This results in a compensation by the counter flow of H3O+ ions towards the cathode: electroendosmosis. the stabilizing medium (e. they can not migrate.g. carboxylic groups in starch and agarose. silicium oxide on glass surfaces. . tubes or capillaries can carry charged groups: e. As they are fixed in the matrix. sulfonic groups in agarose.Principal of electro-endosmosis • The static support.

this effect is observed as a water flow towards the cathode. electroosmotic flow is directed towards the anode. The electrophoretic and electroosmotic migrations are then additive.• In gels. The results are: blurred zones. which carries the solubilized substances along. • When fixed groups are positively charged. and drying of the gel in the anodal area of flatbed gels. the .

• 2.Power back which supply DC. plastic spacers.Instrumentation • 1. .Electrophoresis unit (for running either horizontal or vertical ) : two glass plates (different pore sizes). buffer and comb.

TYPES OF ELECTROPHORESIS ACCORDING TO THE TYPES OF SUPPORT MEDIUM ELECTROPHORESIS FREE ELECTROPHORESIS ZONE ELECTROPHORESIS MICRO ELECTROPHORESIS MOVING BOUNDARY PAPER CELLULOSE ACTTATE GEL ELECTROPHORESIS ELECTROPHORESIS ELECTROPHORESIS .

cellulose acetate filter paper or chromatography paper. This technique is useful for separation of small charged molecules such as amino acids and small proteins.PAPER ELECTROPHORESIS It is the form of electrophoresis that is carried out on filter paper. . FILTER PAPER : It is the stabilizing medium. We can use Whatman filter paper.

Cellulose acetate electrophoresis • One of the older methods but it still has a number of applications. • It gives better resolution than paper electrophoresis . with uniform pore size.however nothing as good as that achieved with polyacrylamide gels. • It has an advantage over paper in that it is a much more homogeneous medium. • It is especially useful for separating alpha immunoglobulins from albumin. . and does not absorb proteins in the way that paper does.

 What is a gel? Gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight and porosity of the target molecules.Polyacrylamide gel. Ribonucleic acid or protein molecules according to their size and electrical charge using an electric current applied to a gel matrix.Agarose gel.GEL ELECTROPHORESIS It is a technique used for the separation of Deoxyribonucleic acid.Starch gel  2.  3. . Types of Gel: 1.

Percentage used (3-30%). Percentage used (1-3%).  Agarose gel :A highly purified uncharged polysaccharide derived from agar.  Used to separate macromolecules such as nucleic acids. .  Polyacrylamide gel (PAGE) : Used to separate most proteins and small oligonucleotides because of the presence of small pores. large proteins and protein complexes. Starch gel : An early form of gel .

An estimation of PCR products sizes was determined by using DNA 100 bp marker in the second lane. Bands in the first lane and third lane are referring to PCR product before and after PCR purification respectively. Both products were DNA electrophoresed in 3% Agarose gel. .Example: PCR products of the C-terminus of killifish CFTR gene before and after PCR purification.

. and sucrose or glycerol. gradient gel and IFE gel. Other used gels are: native gel. PROCEDURE  Protein sample is first boiled for 5 mins in a buffer solution containing SDS and β-mercaptoethanol. and 2D-polyacrylamide gel electrophoresis. cellulose acetate elect.  Before the sample is loaded into the main separating gel a stacking gel is poured on top of the separating gel.  Sample buffer contains bromophenol blue which is used as a tracking dye.  Protein gets denatured and opens up into rod-shaped structure.Sodium dodecyle sulphate (SDS) polyacrylamide gel electrophoresis. The most commonly used technique for the separation of proteins is Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE).

 Blue colored bands are observed under UV rays.  When the dye reaches the bottom of the gel. . owing to the molecular sieving properties of the gel. the current is turned off.  Gel is removed from between the glass plates and shaken in an appropriate stain solution.Cont.  As they pass through the separating gel.  The negatively charged protein-SDS complexes now continue to move towards the anode. the proteins separate.  Current is switched on.

Example The Purification check of the C-terminus of Killifish CFTR. The lanes from (2-5) refer to IMAC unbound materials. Purification was checked using 15% SDS-PAGE gel. The arrow in lane 6 refers to the purified c-terminus peptide. washes and elutions after thrombin cleavage. . washes and elutions before thrombin cleavage while lanes from (6-9) refer to IMAC unbound materials. The Prestained Protein Marker in the first lane was used for molecular weight determinations.

TWO-DIMENSIONAL ELECTROPHORESIS • This technique combines the technique IEF (first dimension). with the size separation technique of SDS-PAGE second dimension). which separates proteins in a mixture according to charge (PI). • The combination of these two technique to give two- dimension(2-D)PAGE provides a highly sophisticated analytical method for analysing protein mixtures. .

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. • B. • 2.second antibodies with marker and then expose blot to UV light. • C. incubation .Proteins: • A.Detection of components after electrophoretic separation • 1.Comassie Brilliant blue.Instant blue. when the current is passed it cause separating of protein from gel to nitrocellulose sheet which will examine further by using antibodies (specific to tested protein) .Silver protein.protein (western blotting): • It involves transferring of proteins from gel to nitrocellulose paper to produce a sandwich of gel and nitrocellulose that compressed in a cassette and immersed in a buffer between parallel electrodes.

Nucleic acids • Ethidium Bromide or SYBR gold . Glycoproteins • Schiffs Reagent (Neutral Glycoprotein). Lipid : • Sudan black • 4. Enzymes • Appropriate enzymatic methods • 5.• 2. • Alcian blue (Acidic mucopolysaccharides) • 3.

metal ions and drugs. • It is a type of zone electrophoresis. peptides. • It involves electrophoresis of sample in a very narrowbore tubes. DNA fragments .Capillary electrophoresis • Used to separate wide range of biological moleculed including : A. proteins.A . • It is a highly speed analysis (20 mins ). .

Microchip electrophoresis • Very high speed analysis (tens of seconds). • Use laser – induced fluorescence. . • It required very low volts.

5. 3. • 2.Use of western blot tech.Identification and quantification specific serum protein • • • • classes. ./ southern blot tech. 6.Clinical applications • 1. 4.Separation and quantification of immunoglobulins.Separation and quantification of enzymes to its subtypes.Specific protein electrophoresis.Separation and quantification of lipoprotein and lipid classes.