BCH 415 LECTURE NOTE

BCH 415: ADVANCED BIOCHEMICAL METHODS (3 UNITS) L:O P:3 T:0

COURSE CONTENT

1. Production, Isolation, Purification and Characterisation of Proteins

2. The Effective Use of The Library

PRODUCTION, ISOLATION, PURIFICATION AND CHARACTERISATION OF

PROTEINS

INTRODUCTION

The sources of proteins are plant, animal, or microbial cells. Certain tissues such as pancreas
glands or liver are particularly rich in enzymes. However, some of the enzymes in the
mixture when cells are disrupted are proteases. They digest proteins. Enzymes are proteins,
and proteases can digest themselves. We can take advantage of the change in enzyme activity
with pH by changing to a pH where the protease activity is low. The disruption of cells to
release enzymes can be carried out at low pH and cold temperatures to minimize losses due to
proteolytic activity.

QUESTION – Why do we always use buffer when working with proteins?

Proteins have great specificity that can be used to attach them to other molecules. Attaching a
protein to a solid by exploiting this specificity is called affinity chromatography. Sometimes
quite pure enzymes can be recovered from a mixture by affinity chromatography, but more
often there is a preliminary separation. Each binding site can attach to only one enzyme
molecule, so it can be expensive to provide enough of the binding agent.

A very common preliminary separation comes from adding ammonium sulfate in stages.
Different proteins precipitate at different salt concentrations, and this divides the enzymes
and other proteins into fractions. There are other salts for precipitation, and polyethylene
glycol is an effective organic precipitant.

The fraction with the desired enzyme also contains much salt. Enzymes form colloidal
suspensions rather than true solutions, but we make the common mistake of saying that the
enzyme is "redissolved" in water. Dialysis with membranes through which the enzyme
cannot pass is the popular method for removing the salt.

Chromatographic separation is covered in much detail in other pages and will not be
discussed here. New techniques such as displacement chromatography can actually
concentrate the enzyme, but conventional chromatography that flushes it through the column
causes much dilution. After concentration by means such as forcing the water out through a
finely pored membrane, freeze drying is a popular method for producing a stable product.
 STEPS IN ISOLATION AND PURIFICATION OF PROTEINS

OBTAIN CELLS BY FERMENTATION OR PURCHASE OF PLANT OR ANIMAL

ORGAN

RELEASE PROTEINS FROM CELLS (RUPTURE BY SHEAR, HOMOGENIZATION,

SONICATION ETC.)

REMOVE CELL DEBRIS (BY FILTRATION OR CENTRIFUGATION)

PRECIPITATE PROTEINS (WITH AMMONIUM SULPHATE OR POLYETHYLENE

GLYCOL)

COLLECT PRECIPITATE AND RESUSPEND FOR SALT REMOVAL (BY DIALYSIS

OR ULTRAFILTRATION)

PURIFICATION (BY CHROMATOGRAPHY)

CONCENTRATE (BY DIALYSIS OR ULTRAFILTERATION) IF DILUTED BY

CHROMATOGRAPY

FREEZE DRYING

QUESTIONS –

1. What is the advantage of Affinity Chromatography

2. Why do we always use buffer when working with proteins?

METHODS OF PROTEIN PURIFICATION

The methods used in protein purification can roughly be divided into analytical and
preparative methods. The distinction is not exact, but the deciding factor is the amount of
protein that can practically be purified with that method. Analytical methods aim to detect
and identify a protein in a mixture, whereas preparative methods aim to produce large
quantities of the protein for other purposes, such as structural biology or industrial use. In
general, the preparative methods can be used in analytical applications, but not the other way
around.

QUESTION – The methods used in protein purification can roughly be divided into
analytical and preparative methods. What determines the choice of method during
protein purification?

EXTRACTION

Depending on the source, the protein has to be brought into solution by breaking the tissue or
cells containing it. There are several methods to achieve this: Repeated freezing and thawing,
sonication, homogenization by high pressure, filtration, or permeabilization by organic
solvents. The method of choice depends on how fragile the protein is and how sturdy the cells
are. After this extraction process soluble proteins will be in the solvent, and can be separated
from cell membranes, DNA etc. by centrifugation. The extraction process also extracts
proteases, which will start digesting the proteins in the solution. If the protein is sensitive to
proteolysis, it is usually desirable to proceed quickly, and keep the extract cooled, to slow
down proteolysis.

QUESTION – There are several methods that can be used in protein extraction. (a)
Enumerate them. (b)What determines the choice of method?

PRECIPITATION AND DIFFERENTIAL SOLUBILIZATION

In bulk protein purification, a common first step to isolate proteins is precipitation with
ammonium sulfate (NH4)2SO4. This is performed by adding increasing amounts of
ammonium sulfate and collecting the different fractions of precipitate protein. Ammonium
sulphate can be removed by dialysis. The hydrophobic groups on the proteins gets exposed to
the atmosphere and it attracts other protein hydrophobic groups and gets aggregated. Protein
precipitated will be large enough to be visible. One advantage of this method is that it can be
performed inexpensively with very large volumes.

The first proteins to be purified are water-soluble proteins. Purification of integral membrane
proteins requires disruption of the cell membrane in order to isolate any one particular protein
from others that are in the same membrane compartment. Sometimes a particular membrane
fraction can be isolated first, such as isolating mitochondria from cells before purifying a
protein located in a mitochondrial membrane. A detergent such as sodium dodecyl sulfate
(SDS) can be used to dissolve cell membranes and keep membrane proteins in solution
during purification; however, because SDS causes denaturation, milder detergents such as
Triton X-100 or CHAPS can be used to retain the protein's native conformation during
complete purification.

QUESTIONS –

1. Download the Ammonium Sulphate Table for Protein Precipitation.

2. Purification of integral membrane proteins requires disruption of the cell membrane in
order to isolate any one particular protein from others that are in the same membrane
compartment. Name the detergents that can be used for this.
3. Which detergent is preferred and why?
4. What is Sonication?

ULTRACENTRIFUGATION

Centrifugation is a process that uses centrifugal force to separate mixtures of particles of

varying masses or densities suspended in a liquid. When a vessel (typically a tube or bottle)
containing a mixture of proteins or other particulate matter, such as bacterial cells, is rotated
at high speeds, the angular momentum yields an outward force to each particle that is
proportional to its mass. The tendency of a given particle to move through the liquid because
of this force is offset by the resistance the liquid exerts on the particle. The net effect of
"spinning" the sample in a centrifuge is that massive, small, and dense particles move
outward faster than less massive particles or particles with more "drag" in the liquid. When
suspensions of particles are "spun" in a centrifuge, a "pellet" may form at the bottom of the
vessel that is enriched for the most massive particles with low drag in the liquid. Non-
compacted particles still remaining mostly in the liquid are called the "supernatant" and can
be removed from the vessel to separate the supernatant from the pellet. The rate of
centrifugation is specified by the angular acceleration applied to the sample, typically
measured in comparison to the g. If samples are centrifuged long enough, the particles in the
vessel will reach equilibrium wherein the particles accumulate specifically at a point in the
vessel where their buoyant density is balanced with centrifugal force. Such an "equilibrium"
centrifugation can allow extensive purification of a given particle.

Sucrose gradient centrifugation — a linear concentration gradient of sugar (typically

sucrose, glycerol, or a silica-based density gradient media, like Percoll) is generated in a tube
such that the highest concentration is on the bottom and lowest on top. Percoll is a trademark
owned by GE Healthcare companies. A protein sample is then layered on top of the gradient
and spun at high speeds in an ultracentrifuge. This causes heavy macromolecules to migrate
towards the bottom of the tube faster than lighter material. During centrifugation in the
absence of sucrose, as particles move farther and farther from the center of rotation, they
experience more and more centrifugal force (the further they move, the faster they move).
The problem with this is that the useful separation range of within the vessel is restricted to a
small observable window. Spinning a sample twice as long doesn't mean the particle of
interest will go twice as far, in fact, it will go significantly further. However, when the
proteins are moving through a sucrose gradient, they encounter liquid of increasing density
and viscosity. A properly designed sucrose gradient will counteract the increasing centrifugal
force so the particles move in close proportion to the time they have been in the centrifugal
field. Samples separated by these gradients are referred to as "rate zonal" centrifugations.
After separating the protein/particles, the gradient is then fractionated and collected.

CHROMATOGRAPHIC METHODS

Chromatographic equipment. Here set up for a size exclusion chromatography. The buffer is
pumped through the column (right) by a computer controlled device.

Usually a protein purification protocol contains one or more chromatographic steps. The
basic procedure in chromatography is to flow the solution containing the protein through a
column packed with various materials. Different proteins interact differently with the column
material, and can thus be separated by the time required to pass the column, or the conditions
required to elute the protein from the column. Usually proteins are detected as they are
coming off the column by their absorbance at 280 nm. Many different chromatographic
methods exist:

1. SIZE EXCLUSION CHROMATOGRAPHY

Gel filtration chromatography is based on the molecular size and the hydrodynamic volume
of the components. The molecules are separated by the differential exclusion or inclusion of
solutes as they pass through the stationary phase. The stationary phase used is a porous
polymer matrix whose pores are completely filled with the solvent to be used as the mobile
phase. The molecules in th e sample are pumped through specialized columns containing
heteroporous/microporous cross-linked polymeric gel or beads. The flow of the mobile phase
will separate the components of the sample based on their sizes. The basis of the separation is
that molecules above a certain size are totally excluded from the pores, while smaller
molecules access the interior of the pores partly or wholly. Large molecules are entirely
excluded from the pores and come first in the effluent whereas smaller molecules will be
retarded according to their penetration of the gel. Smaller molecules get distributed between
the mobile phase, inside and outside of the pores of the gel. Then, they pass through the
column at a slower rate and appear later in the effluent

Chromatography can be used to separate protein in solution or denaturing conditions by using

porous gels. This technique is known as size exclusion chromatography. The principle is that
smaller molecules have to traverse a larger volume in a porous matrix. Consequentially,
proteins of a certain range in size will require a variable volume of eluent (solvent) before
being collected at the other end of the column of gel.

In the context of protein purification, the eluant is usually pooled in different test tubes. All
test tubes containing no measurable trace of the protein to purify are discarded. The
remaining solution is thus made of the protein to purify and any other similarly-sized
proteins.

2. SEPARATION BASED ON CHARGE OR HYDROPHOBICITY

(a) Hydrophobic Interaction Chromatography

Resin used in the column are amphiphiles with both hydrophobic and hydrophilic regions.
The hydrophobic part of the resin attracts hydrophobic region on the proteins. The greater the
hydrophobic region on the protein the stronger the attraction between the gel and that
particular protein.

(b) Ion exchange chromatography

Ion exchange chromatography separates compounds according to the nature and degree of
their ionic charge. The column to be used is selected according to its type and strength of
charge. Anion exchange resins have a positive charge and are used to retain and separate
negatively charged compounds, while cation exchange resins have a negative charge and are
used to separate positively charged molecules.

Before the separation begins a buffer is pumped through the column to equilibrate the
opposing charged ions. Upon injection of the sample, solute molecules will exchange with
the buffer ions as each competes for the binding sites on the resin. The length of retention for
each solute depends upon the strength of its charge. The most weakly charged compounds
will elute first, followed by those with successively stronger charges. Because of the nature of
the separating mechanism, pH, buffer type, buffer concentration, and temperature all play
important roles in controlling the separation.

Ion exchange chromatography is a very powerful tool for use in protein purification and is
frequently used in both analytical and preparative separations.

(c) Affinity chromatography

Affinity Chromatography is a separation technique based upon molecular conformation,

which frequently utilizes application specific resins. These resins have ligands attached to
their surfaces which are specific for the compounds to be separated. Most frequently, these
ligands function in a fashion similar to that of antibody-antigen interactions. This "lock and
key" fit between the ligand and its target compound makes it highly specific, frequently
generating a single peak, while all else in the sample is unretained.

Many membrane proteins are glycoproteins and can be purified by lectin affinity
chromatography. Detergent-solubilized proteins can be allowed to bind to a chromatography
resin that has been modified to have a covalently attached lectin. Proteins that do not bind to
the lectin are washed away and then specifically bound glycoproteins can be eluted by adding
a high concentration of a sugar that competes with the bound glycoproteins at the lectin
binding site. Some lectins have high affinity binding to oligosaccharides of glycoproteins that
is hard to compete with sugars, and bound glycoproteins need to be released by denaturing
the lectin.

Fig. Nickel-affinity column. The resin is blue since it has bound nickel.

Metal binding

A common technique involves engineering a sequence of 6 to 8 histidines into the N- or C-

terminal of the protein. The polyhistidine binds strongly to divalent metal ions such as nickel
and cobalt. The protein can be passed through a column containing immobilized nickel ions,
which binds the polyhistidine tag. All untagged proteins pass through the column. The
protein can be eluted with imidazole, which competes with the polyhistidine tag for binding
to the column, or by a decrease in pH (typically to 4.5), which decreases the affinity of the
tag for the resin. While this procedure is generally used for the purification of recombinant
proteins with an engineered affinity tag (such as a 6xHis tag or Clontech's HAT tag), it can
also be used for natural proteins with an inherent affinity for divalent cations.

Immunoaffinity chromatography

A HPLC. From left to right: A pumping device generating a gradient of two different
solvents, a steel enforced column and an apparatus for measuring the absorbance.

Immunoaffinity chromatography uses the specific binding of an antibody to the target protein
to selectively purify the protein. The procedure involves immobilizing an antibody to a
column material, which then selectively binds the protein, while everything else flows
through. The protein can be eluted by changing the pH or the salinity. Because this method
does not involve engineering in a tag, it can be used for proteins from natural sources.

Purification of a tagged protein

Adding a tag to the protein such as RuBPS gives the protein a binding affinity it would not
otherwise have. Usually the recombinant protein is the only protein in the mixture with this
affinity, which aids in separation. The most common tag is the Histidine-tag (His-tag), that
has affinity towards nickel or cobalt ions. Thus by immobilizing nickel or cobalt ions on a
resin, an affinity support that specifically binds to histidine-tagged proteins can be created.
Since the protein is the only component with a His-tag, all other proteins will pass through
the column, and leave the His-tagged protein bound to the resin. The protein is released from
the column in a process called elution, which in this case involves adding imidazole, to
compete with the His-tags for nickel binding, as it has a ring structure similar to histidine.
The protein of interest is now the major protein component in the eluted mixture, and can
easily be separated from any minor unwanted contaminants by a second step of purification,
such as size exclusion chromatography or RP-HPLC.

Another way to tag proteins is to engineer an antigen peptide tag onto the protein, and then
purify the protein on a column or by incubating with a loose resin that is coated with an
immobilized antibody. This particular procedure is known as immunoprecipitation.
Immunoprecipitation is quite capable of generating an extremely specific interaction which
usually results in binding only the desired protein. The purified tagged proteins can then
easily be separated from the other proteins in solution and later eluted back into clean
solution.0

When the tags are not needed anymore, they can be cleaved off by a protease. This often
involves engineering a protease cleavage site between the tag and the protein.

3. HPLC
High performance liquid chromatography or high pressure liquid chromatography is a form
of chromatography applying high pressure to drive the solutes through the column faster.
This means that the diffusion is limited and the resolution is improved. The most common
form is "reversed phase" hplc, where the column material is hydrophobic. The proteins are
eluted by a gradient of increasing amounts of an organic solvent, such as acetonitrile. The
proteins elute according to their hydrophobicity. After purification by HPLC the protein is in
a solution that only contains volatile compounds, and can easily be lyophilized.[3] HPLC
purification frequently results in denaturation of the purified proteins and is thus not
applicable to proteins that do not spontaneously refold.

Concentration of the purified protein

A selectively permeable membrane can be mounted in a centrifuge tube. The buffer is forced
through the membrane by centrifugation, leaving the protein in the upper chamber.

At the end of a protein purification, the protein often has to be concentrated. Different
methods exist.

Lyophilization

If the solution doesn't contain any other soluble component than the protein in question the
protein can be lyophilized (dried). This is commonly done after an HPLC run. This simply
removes all volatile component leaving the proteins behind.

Ultrafiltration

Ultrafiltration concentrates a protein solution using selective permeable membranes. The

function of the membrane is to let the water and small molecules pass through while retaining
the protein. The solution is forced against the membrane by mechanical pump or gas pressure
or centrifugation.

Analytical

Denaturing-Condition Electrophoresis

Gel electrophoresis is a common laboratory technique that can be used both as preparative
and analytical method. The principle of electrophoresis relies on the movement of a charged
ion in an electric field. In practice, the proteins are denatured in a solution containing a
detergent (SDS). In these conditions, the proteins are unfolded and coated with negatively
charged detergent molecules. The proteins in SDS-PAGE are separated on the sole basis of
their size.

In analytical methods, the protein migrate as bands based on size. Each band can be detected
using stains such as Coomassie blue dye or silver stain. Preparative methods to purify large
amounts of protein, require the extraction of the protein from the electrophoretic gel. This
extraction may involve excision of the gel containing a band, or eluting the band directly off
the gel as it runs off the end of the gel.
In the context of a purification strategy, denaturing condition electrophoresis provides an
improved resolution over size exclusion chromatography, but does not scale to large quantity
of proteins in a sample as well as the late chromatography columns.

Non-denaturing gel electrophoresis (non SDS-PAGE, native PAGE)

The SDS_PAGE technique described above is the commonest method used for
electrophoretic separation of proteins. In some situations, however, proteins may be resolved
on so-called “native” gels, in the absence of SDS. Under these conditions, the movement of
proteins through the gel will be affected not simply by their mass, but by their charge at the
pH of the gel, as well. Proteins complexed with other molecules may move as single entity,
allowing the isolation of the binding partners of proteins of interest.

What is the difference between non SDS-PAGE and SDS-PAGE?

The major difference between native PAGE and SDS-PAGE is that in native PAGE, the
protein migration rate is dependent on both the mass and structure, whereas in SDS-PAGE,
the migration rate is determined only by protein’s mass.

In native PAGE, protein samples are prepared in a non-denaturing and non-reducing buffer.
Therefore, in addition to the size of protein, the secondary, tertiary as well as quaternary
structure all has an impact on the migration.

In SDS-PAGE, an excessive amount of SDS is added to coat proteins in negative charge,

which covers all the intrinsic charges of protein. A similar charge-to-mass ratio is obtained
for all proteins, and the electrophoretic mobility in SDS-PAGE depends almost solely on the
size of protein.

Non-Denaturing-Condition Electrophoresis

The understanding of processes in living systems depends to a great extent on our ability to
isolate bioactive compounds (e.g., proteins) in biological samples for more detailed
examination of chemical structure and function. As about 30-40 % of all known proteins
contain metal cofactors (e.g., Fe, Cu, Zn, Mo, Ni), especially native metalloproteins have to
be isolated, identified and quantified in biomatrices. Many of these cofactors play a key role
in enzymatic catalytic processes or stabilize globular protein molecules.

An important non-denaturing electrophoretic procedure for isolating bioactive

metalloproteins in complex protein mixtures is termed 'quantitative native continuous
polyacrylamide gel electrophoresis (QPNC-PAGE). QPNC-PAGE, or quantitative
preparative native continuous polyacrylamide gel electrophoresis, is a high-resolution
technique applied in biochemistry and bioinorganic chemistry to separate proteins by
isoelectric point. This variant of gel electrophoresis is used by biologists to isolate active or
native metalloproteins in biological samples and to resolve properly and improperly folded
metal cofactor-containing proteins in complex protein mixtures.
QUESTIONS ON CHROMATOGRAPHY

1. What is chromatography?

2. What is the basis (principle) of chromatographic process?

3. What type of solvents are generally employed in chromatography?

4. Name some chromatographic techniques.

5. What is meant by the term Rf value?

6. On what factors does the Rf value of a compound depend?

7. Give the biochemical uses of chromatography.

8. What are the advantages of chromatography over other techniques?

9. What is the principle involved in column chromatography?

10. With a named column chromatography, write a short note on Chromatography

Techniques.

QUESTIONS ON PROTEIN SEPARATION

1. How will you obtain the crude extract of a protein?

2. Enumerate the steps involved in purifying proteins to homogeneity.

3. Explain the steps involved in purifying proteins to homogeneity.

4. Write short notes on 5 chromatographic methods that can be used in separating

proteins.

CHARACTERIZATION OF PROTEIN

A complete characterization of a given protein may involve the use of multiple techniques. A
thorough characterization of the studied protein can be achieved by the quantitative
determination of the protein concentration, for example by using amino acid analysis or, the
measuring of the absorbance of the protein in solution at 280 nm using an ultraviolet (UV)
spectrophotometer. The intact molecular weight can be measured using mass spectrometry.
Next, the protein sequence can be elucidated using N-terminal Edman chemistry based
protein sequencing, and N- and C-terminal sequencing by mass spectrometry. Additional
techniques for further characterizations are peptide mapping using chemical or enzymatic
digest in combination with reversed phase liquid chromatography and tandem mass
spectrometry. For this, often the protein has first to be isolated or separated from other
proteins, metabolites or impurities present in the source. Routine techniques such as
polyacrylamide based gel electrophoresis (1D or 2D PAGE) in combination with blotting
techniques (Electro-blotting or Western blotting) are used. Alternatively, liquid
chromatographic methods (1D or 2D, ion exchange, reversed phase, etc.) may offer a more
detailed analysis.

Fig. Protein identification and characterization

Estimation of protein molecular weight

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophores): the most widely
used technique to separate proteins from complicated samples of mixture, used in protein
identification including estimations of proteins molecular weight, identification, purification
and assessment of purity.

Western Blot (also known as immunoblot or protein blot): a widely used analytical technique
used to detect specific protein in a complex mixture extracted from cells while giving you
information about the size of target protein, dependent on the use of a high-quality antibody
directed against the protein. The capability to clearly show the presence of a specific protein
both by size and through the binding of an antibody makes it well-suited for evaluating levels
of protein expression in cells, and for monitoring fractions during protein purification.
Likewise, it is helpful for comparing expression of a target protein from various tissues, or
seeing how a particular protein responds to disease or drug treatment.

Assessment of Protein Purity

SDS-PAGE a commonly used protein identification technique, based on the difference

between protein molecular weight to assess protein purity.
HPLC (High-Performance Liquid Chromatography): a form of column chromatography that
pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure
through a column with chromatographic packing material (stationary phase). HPLC has the
capability to separate, identify, and quantify each components that are present in any sample
that can be dissolved in a liquid in trace concentrations as low as parts per trillion. Because of
this versatility, HPLC is used in a variety of industrial and scientific applications, such as
pharmaceutical industry, environmental monitoring and chemical industry.

Determination of Protein Concentration

Bradford assay: Bradford assay is a colorimetric assay based on the interaction between
Coomassie brilliant blue and arginine and aromatic residues in your protein, resulting in a
shift of the absorption maximum of the dye from 465 to 595 nm. In general, the absorbance
of a series of known concentrations of a standard protein, generally BSA, is measured to
create a standard curve, which can be used to calculate the concentration of your protein
sample based on its absorbance.

BCA assay: BCA, another protein analysis assay, makes use of biuret reaction, in which
protein backbone chelates Cu2+ ions and reduces them to Cu1+ ions. The Cu1+ ions then
react with bicinchoninic acid (BCA) to form a purple-colored product that absorbs at 562 nm.
The procedure is similar to that of the Bradford assay, in which you create a standard curve
based on a series of known protein standards.

ELISA (Enzyme-Linked Immunosorbent Assay): also known as an enzyme immunoassay

(EIA), is a common laboratory technique designed for detecting and quantifying an analyte
(usually antibodies or antigens). In an ELISA test, an antigen is immobilized to a solid
surface and then the antigen combines with an antibody that is linked to an enzyme.
Detection is accomplished by assessing conjugated enzyme activity via incubation with a
substrate to produce a measureable product. ELISA is quick and simple to carry out, and
since it is designed to rapidly handle a large numbers of samples in parallel, it is a very
popular choice for evaluation of various research and diagnostic targets.

Protein Identification by Mass Spectrometry

Mass spectrometry is an important emerging method for protein identification and

characterization. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization
(MALDI) are two primary methods for ionization of whole proteins. In keeping with the
performance and mass range of available mass spectrometers, two approaches are used for
characterizing proteins including "top-down" strategy and "bottom-up"strategy. In "top-
down" strategy of protein analysis, intact proteins are ionized by either of the two techniques
described above, and then introduced to a mass analyzer. In "bottom-up" proteomics,
identification of the existence of proteins is at the peptide level. A common procedure of
"bottom-up" strategy of protein analysis involves using tryptic digestion to obtain masses of
individual peptides derived from the protein. Subsequently these peptides are introduced into
the mass spectrometer and identified by peptide mass fingerprinting or tandem mass
spectrometry. Then the masses are compared against an online database, and probability-
based scoring systems are used to determine the closest protein matches.

Amino Terminal Sequence

Amino-terminal (N-terminal) sequence analysis is used to identify the order of amino acids of
proteins or peptides, starting at their N-terminal end. The composition of N-terminal
sequence has a huge impact on the biological functions and stability of proteins, for example
the half-life, subcellular localization, post-translational modification of proteins. Analysis of
N-terminal sequence contributes to protein senior structure analysis, further revealing
biological function of proteins. BIC provides N-terminal sequence analysis by both Edman
degradation and mass spectrometry.

Evaluation of Protein Stability

Protein stability evaluation is often considered as an important component of protein

production since protein targets can be challenging to work with due to their susceptibility to
degradation and aggregation. Protein freezing and thawing assay can be performed to
examine protein stability in the process of transport, storage and applications under the
condition of special circumstances.

Detection of Endotoxin Level

Endotoxin is a major contaminant found in biologically active substances affecting both

invitro and invivo cell growth and function. Thorough cleanliness in lab ware, raw materials,
and in lab technique is required to substantially reduce endotoxin levels. This is achieved by
gel clot assay.

Analysis of Protein Structure

Circular Dichroism (CD) is based on measuring the differential absorption of left- and right-
handed circularly polarised light by optical active compounds at different wave lengths. CD
spectroscopy is a sensitive tool to study secondary structure changes and provides the basis
for the conformational analysis of macromolecules. The method can be used for the
determination of the protein secondary structure (α-helix, β-structure, β-turns) and nucleic
acid conformation. Another application of CD spectroscopy is the examination of
protein/nucleic acid stability (folding, unfolding and refolding) under the effect of different
factors (pH, denaturants, temperature) and the calculation of thermodynamic parameters
related to these processes.

QUESTIONS

1. Enumerate 5 methods of characterisation of proteins

2. A complete characterization of a given protein may involve the use of multiple

techniques. Explain.
 THE EFFECTIVE USE OF THE LIBRARY
WHAT IS A LIBRARY?
Traditionally, a library is defined as physical space (building) emphasizing physical (print)
collections. Emphasis is on storage and preservation of physical items particularly books and
periodicals. Information is physically assembled in one place and users must visit the library
to use the materials. However, the introduction of ICT, which brought about the introduction
of new formats of information, has given the concept of library a new definition. It is now
defined as: A place where information resources are accessed and information services
are rendered by professionals who specialize in identifying, collecting, organizing,
processing information sources as well as interpreting information needs.
The word ‘place’ could be physical or virtual. Physical place refers to a library building or
library collection. The concept of a virtual library place emphasizes on remote access to
digital information resources and services through computer terminals.

TYPES OF LIBRARY
There are different types of libraries. Categorization is based on the parent institution that
established them, type of materials they manage, the subject matter of the materials and users
of the library. Hence, libraries are categorized into:
1. Academic libraries – Academic libraries are those libraries attached to institutions of
higher learning of the status of a tertiary institution. In other words, academic libraries
are found in post-secondary institutions such as Universities, polytechnics and
colleges of education. Academic libraries are primarily established to provide
literature support to the programmes of their parent institution, to aid lecturers,
students and researchers in teaching, learning, research and recreation purposes. For
instance, if a tertiary institution is offering Degree, Diploma and Certificate courses,
its library is expected to provide educational, research and information materials in
the relevant subjects to suit each level of the institution’s academic programmes.
2. School Libraries – These are libraries established in primary and secondary schools
3. Public libraries – These are libraries established and funded by government to serve
members of the general public.
4. Research libraries – These are libraries established and funded by research institutes
to provide collections that are specific to the research focus of their parent institutions.
5. Special libraries – These are libraries owned by private or public institutions that
render services in specialized fields of the society. Examples are law libraries,
hospital libraries, church etc.

FUNCTIONS UNITS OF A LIBRARY

The library has several Units that work together to collect, receive, process and deliver
information products and services. They are categorized into three namely: administrative,
technical services and readers’ services units.
Administrative Unit – It plans, organizes, leads and controls the activities of the library and it
is headed by the University Librarian.
Technical Services Unit – has sub-units which are: acquisitions, cataloguing and
classification, serials and documents and systems unit. –
Acquisition unit– responsible for the selection, ordering and acquisition of library materials
Cataloguing and classification unit – processes acquired materials.
Serials unit - this unit selects, orders, receives, processes and shelves serials publications.
The readers’ services unit/public services unit – in this unit, patrons/users have direct
relationship or contact with the library. It comprises of circulation and reference sub-units.
Circulation sub-unit – responsible for charging and discharging books to patrons/users.
Charging and discharging means books are lent out to (charging) and returned by
(discharging) patrons/users as and when due.
- It registers new users and serves as inquiry desk for library visitors.
- It keeps records and statistics of users and library holdings
- Keeps library management abreast of the state of the library collections
- It handles and curbs all forms of delinquent practices and difficult user behaviours.
Reference sub-unit – As the name implies, the materials in this unit are for reference only.
- The unit exists to support users to find, access, retrieve, evaluate and use information.
Main services are:
- Current awareness
- Selective dissemination of information (SDI)
- Compilation of reading lists and subject guides
- Library orientation and information literacy training.

CATEGORIES OF LIBRARY STAFF

 Librarians
 Library Officers
 Library Assistants
 Library Porters

TYPES OF LIBRARY MATERIALS

Library materials, also known as library stock, can be grouped under two sub-topics namely:
1. Books
2. Non-book materials.
Library materials are not limited to books as it is assumed in some quarters. The break-down
is as follows:
(1.) BOOK MATERIALS: Book materials are those materials in printed form that can be
read and understood by the readers. Thus, a book can be defined as a number of printed or
written pages of not less than 49pages, bound together along one edge and usually protected
by either hardback or paperback cover. When it is less than 49 pages, it is not a book, but a
pamphlet or a booklet. The list of book materials includes:
 TEXTBOOKS —These are books on different subject areas (disciplines) also known
as non-fiction materials while literature books are the fiction materials, such as novels
or short stories which are literary works, invented by imagination.
 REFERENCE MATERIALS — These are books that provide clue to reference
queries (questions), such as: What? Which? How? Why? etc. Reference books
(materials) are: Dictionaries, Encyclopaedias, Directories, Yearbooks, Handbooks,
Gezettes, Gazetteers, Indexes, Atlases, Almanacs, Bibliographies, Biographies etc
Reference books are not meant to be read from page to page, cover to cover like other
books. Rather, reference books provide answer to questions, terms or terminologies
and they are usually alphabetically arranged, and are mostly in volumes.
 PERIODICALS — These are publications which are issued at regular or an irregular
intervals, usually with volume and date, with the intention of being continued
indefinitely, which could be daily, weekly, monthly, bi-annually or annually.
Examples are Daily Newspapers (Dailies), Weekly Magazines, Journals, Theses and
Dissertations, Reports, Past question papers etc.
(2). NON-BOOK MATERIALS: Non-book materials are variously called: -visual materials
-print media or Non-book media. Non-book materials help in solving communication
problem and enhance instructional efficiency during teaching and learning process, as it
enables students to see those things being taught in real life situations, as seeing is believing.

ORGANIZATION OF LIBRARY RESOURCES

A library is a collection of sources, resources, and services, and the structure in which it is
housed. A library is a collection of useful materials for common use. However, with the sets
and collection of media and other books for storing information, many libraries are now also
repositories and access points for maps, prints, or other documents and various storage media
such as microform, audio tapes, CDs, cassettes, videotapes, DVDs, and video games. Modern
libraries are increasingly being redefined as places to get unrestricted access to information in
many formats and from many sources. They are understood as extending beyond the physical
walls of a building, by including material accessible by electronic means, and by providing
the assistance of librarians in navigating and analyzing tremendous amounts of information
with a variety of digital tools. (Wikipedia, 2010) Libraries also provide facilities to access
subscription databases and the Internet. Academic libraries differ from each other in many
respects but they all have the same basic function, which is to aid the parent institution in
carrying out its objectives in the areas of teaching, research and community development.
The library contributes to the realization of these objectives and supports the total programme
by acquiring and making available the books, materials, services that are needed.
LIBRARY COLLECTIONS: In order to make these resources more easily accessible and
retrievable, library resources are organized into various collection using online catalogues.
Online cataloguing has greatly enhanced the usability of catalogues, thanks to the Machine
Readable Cataloguing-MARC standard in the 1960s. MARC was originally used to automate
the creation of physical catalogue cards; now the MARC computer files are accessed directly
in the search process. Online Public Access Catalogues (OPACs) operations have enhanced
usability over traditional cards formats.
LIBRARY CATALOGUE: The university library information resources are classified and
shelved using the Library of Congress classification scheme. The resources are catalogued
using the internationally recognized conventions such as Anglo America Cataloguing Rules.
The processed bibliographic data is presented in two formats, printed catalogue cards and
automated Online Public Access Catalogues (OPACs). The catalogue is available for
concurrent use by several users of the library. The catalogue cards are stored in a catalogue
cabinet with dimensions and structure that allow quick and easy access by most users. The
cabinet drawers are conspicuously labelled in alphabetical order. To enable a person to find a
book of which either through: the author, the title, the subject. Secondly, to show what the
library has: by a given author, on a given subject and in a given kind of literature.
ONLINE PUBLIC ACCESS CATALOGUES (OPACs): The automated catalogue is
through computers for easy access and retrieval of information in the library. The computers
access points are situated next to the catalogue cabinets to provide users with options of using
either format to access the library collection. The Online Public Access Catalogues (OPACs)
is available in the campus wide information network. The content of library information
resources are made accessible through indexes in printed or computer based formats. The
university library catalogue allows for appropriate editing to keep abreast with modern
technology, contemporary practices and changing international information.

HOW TO ACCESS INFORMATION FROM CATALOGUE

1. Author catalogue: sorted alphabetically to the authors’ or editors’ names of the entries.
2. Title catalogue: sorted alphabetically according to the title of the entries.
3. Dictionary catalogue: a catalogue in which all entries (author, title, subject, and series) are
interfiled in a single alphabetical order.
4. Reference Library catalog: Retrieved from www.wikipedia on 26th July, 2010.

LIBRARY E-RESOURCES AND DATABASES

The university library has lots of e-resources and databases for the use of academic staff and
both undergraduate and postgraduate students. These resources are e-journals, e-books,
databases and internet connectivity. The university library also subscribes to lots of websites
which is available to users at the library. These resources can be accessed at the serials
section and e- library of the university library.
1. Databases are arranged and stored in formats that permit efficient searches. There are
many types of databases- including databases for looking up numerical information, such as
statistics, and bibliographies and holding information on books and periodicals, and databases
that allow the user to search and use academic papers and article from journals and
newspapers, encyclopaedias, dictionaries, and case law digest. Some databases come on CD-
ROMs or DVD-ROMs to be used with a single computer, although today online databases
available over the internet are widely used.
2. E-journals are digital versions of articles from journals that allow you to read the full text
on the internet. The advantages of online e-journals are as follows:
Academic papers can be accessed on the web before publication of the printed edition.
It is easier to find individual academic papers online than in printed edition.
e-journals are accessible anytime from campus terminals and laboratory terminals and outside
the campus.
3. E-books are digital versions of books that allow you to read the full text on the internet. At
the same time, you can download the e-books free. Old literary, scientific work,
encyclopaedias, and dictionaries are available as e-books.
4. Internet based service: Internet contains the biggest resources of information in the entire
world; secondly, it enables users to obtain an interactive mechanism to instantly
communicate with each other. Once connected to the internet, everyone can enjoy the
unparallel richness of global information resources including textual, audio, graphic
information. The internet information resources are constantly expanding at a great speed-one
can only make a rough estimate. The types of information on the internet are also wide
ranging, from scientific research, education, public policy, legal regulations to commerce,
arts and entertainment.

E-RESOURCES COLLECTION OF UNIVERSITY LIBRARY

1. HEALTH INTERNATIONAL NETWORK ACCESS TO RESEARCH INITIATIVES
(HINARI) URL: www.who.int/hinari
2. EBSCOHOST URL: http://search.ebscohost.com User ID: ns083634 Password: password
3. ACCESS TO GLOBAL ONLINE RESEARCH IN AGRICULTURE (AGORA) URL:
http://www.aginternetwork.org/
4. JOINT SYSTEM TO ORDER RESOURCES (JSTOR) URL: www.jstor.org
5. Emerald Journals URL: http://www.emeraldinsight.com Username: NigUnaab Password:
library
6. Popline Journals URL: www.popline.org
7. AFRICAN JOURNAL ONLINE (AJOL) URL: http://ajol.info/
8. BIOLINE INTERNATIONAL URL: http://www.bioline.org.br/journals
9. DIRECTORY OF OPEN ACCESS JOURNALS (DOAJ) URL: http://www.doaj.org/
10. THE ESSENTIAL ELECTRONIC AGRICULTURAL LIBRARY (TEEAL)
11. E-GRANARY DIGITAL LIBRARY
12. NATIONAL VIRTUAL LIBRARY OF NIGERIA URL: www.nigerianvirtuallibrary.com
Username: UNAAB Password: Abeokuta
13. BioOne URL: http://www.bioone.org. Email: bamigboye66@yahoo.com Password:
password Areas of coverage: All disciplines
15. Intuite URL: www.intute.com Areas of coverage: Sciences, Social sciences and Health
education.
16. NIGERIA US EMBASSY URL: http://nigeria.usembassy.gov/
http://nigeria.usembassy.gov/hr_office.html http://nigeria.usembassy.gov/irc.html

E-LIBRARY
Information technology has revolutionized the concept of libraries. Each and every library is
getting digitized. There are many definitions of an e-library; terms such as virtual library,
electronic library and digital library are often used synonymously. The e-library is nothing
but a large database for the researchers who are working on hypertext environment. It is an
environment, which supports the full cycle of creation, storage, preservation, dissemination
and use of data, information and knowledge. According to Arms (2007) an e-library is a
managed collection of information with associated services where the information is stored in
digital format and accessible over a network. An e-library is an organized collection of
digitized material or its holding in the digital form, which can be accessible by a computer on
the network by using TCP/IP or other protocol. The e-library is an organized collection of
multimedia and other types of resources, resources are available in computer processable
form, the function of acquisition, storage, preservation, and retrieval is carried out through
the use of digital technology. Access to the entire collection is globally available directly or
indirectly across a network, supports users in dealing with information objects and helps in
the organization and presentation of the above objects via electronic / digital means etc.
ADVANTAGES: E-library is not confined to a particular location or so called building, it is
virtually distributed all over the world. The user can get his/her information on his own
computer screen by using the internet. Actually, it is a network of multimedia system, which
provides fingertip access.
1. No physical boundary: The user of an e-library need not go to the library physically;
people from all over the world could gain access to the same information, as long as an
internet connection is available.
2. Round the clock availability: E- libraries can be accessed at any time, 24 hours a day and
365days of the year.
3. Multiple accesses: The same resources can be used at the same time by a number of users.
4. Structured approach: E-library provides access to much richer content in a more structured
manner. We can easily move from the catalog to the particular book then to a particular
chapter and so on.
5. Information retrieval: The user is able to use any search term bellowing to the word or
phrase of the entire collection. E-library will provide every user-friendly interface, giving
click able access its resources.
6. Preservation and conservation: An exact copy of the original can be made any number of
times without any degradation in quality.
7. Space: Whereas traditional libraries are limited by storage of space, e-library has the
potential to store much more information, simply because e-information requires very little
physical space to contain them. When the library had no space for extension digitization is
the only solution.
8. Networking: A particular e-library can provide the link to any other resources of other e-
library very easily thus to a seamlessly integrated resource sharing can be achieved.
9. Cost: The cost of maintaining an e-library is much lower than of a traditional library. A
traditional library must spend large sums of money paying for staff, book maintenance. E-
libraries do away with these fees.
DISADVANTAGES: The computer viruses, lack of standardization for digitized
information, quick degrading properties of digitized materials and its associated problem,
health hazard nature of the radiation from monitor etc. makes e-libraries at times handicap.
1. Copyright: Virtualization violates the copyright law as other can freely transfer the thought
content of one author without his acknowledgement. So one difficulty facing e-library is the
way to distribute information. How does an e-library distribute information at will, while
protecting the copyright of the author?
2. Speed of access: As more and more computer are connected to the internet its speed of
access reasonably decreasing. If new technology will not evolve to solve the problem in near
future internet will be full of error messages.
3. Bandwidth: E-library will need high band for transfer of multimedia resources but the band
width is decreasing day by day due to its over utilization.
4. Initial cost is high: The infrastructure cost of an e-library i.e. the cost of hardware,
software, leasing communication circuit is generally very high.
5. Efficiency: With the much larger volume of e-information, finding the right material for a
specific task becomes increasingly difficult.
6. Environment: E-libraries cannot reproduce the environment of a traditional library. Many
researchers still finds reading printed material to be easier than reading material on a
computer screen.

LIBRARY RULES AND REGULATIONS

INTRODUCTION: There are rules and regulations which are meant to guide the use and
conduct of users of the library. It is very important that students should be familiar with such
rules and regulations in order not to violate any of them. The rules and regulations are usually
contained in the Readers’ Guide or Students’ Handbook of some tertiary institutions. These
are to guide the conduct and behaviours of the readers in the library and to assist the library to
achieve its set goals and objectives and for the users to make maximum use of the library
services. Defaulters of these rules and regulations are to be sanctioned (penalized) for it, to
serve as deterrent to others. Some of the rules and regulations are:
1) Possession of identification card when visiting the library
2) Any materials leaving the library must be properly borrowed.
3) Books in the Reference Section, Serials or Reserved Units may not be removed from
the library.
4) It is an offence to keep materials (books) beyond the date specified for return.
5) Penalties (fine) will be charged for over-due books.
6) Returned books must be delivered at the Loans’ Desk.
7) All consulted books must be left on the Reading Tables.
8) No Readers may enter any part of the library marked ‘Private or Work-room’ unless
by permission.
9) Any person who is suspected to be security risk may be ordered out of the library.
10) Indecent dressing will not be allowed into the library.
11) The use of naked light is not allowed in the library.
12) Marking or underlining of library books is not allowed
13) Briefcases, luggage, umbrella, camera etc are not allowed into the library
14) Smoking, eating etc is not allowed in the library
15) Pets must not be brought into the library
16) Silence must be maintained in and around the library
17) Only registered users are allowed to use the library resources
18) Users must present whatever materials they are carrying to the security personnel at
the entrance for checking while leaving the library.
 19) The use of cell phone is prohibited in the library
20) Reservation of seat in the library is prohibited
21) Book mutilation, pilfering, theft are all prohibited

RATIONALE FOR THIS COURSE

1. The library provides different information resources and services geared the
enhancement of academic achievement.
2. To efficiently and effectively exploit the library resources and service, it is important
for students to acquire some knowledge of the working of the library, learn skills
necessary for locating, sifting, evaluating and sorting information sources.
3. The course is designed to enable students acquire the required skills.
4. The skills are relevant in writing research project, conference papers, journal articles
and contribution to books.

PRIMARY USES OF THE LIBRARY

People use the Library, primarily for three purposes:
1. To get to know it as a general purpose resource they can use over time.
2. To find a type of resource to solve a current problem, achieve a goal or meet an
interest.
3. To learn how various topics can conventionally be categorized and integrated with
each other.

References
1) Arms, L.C. (2007). Virtual Library: Needs, Technology and Benefit. ILA Bulletin,
38(3),pp22-26
2) E-library and resources. (2010) Retrieved from
http://en.wikipedia.org/wiki/digitallibrary.
3) Okeh, E.G.ed. (2008). Understanding the Use of the Library and Information
Services. Ibadan: Euniprints International Media.
4) Imam, A., Adeyoyin, S.A., Jegede, O.R. & Adesanya, O. (2008). Library and
Information Studies: An Introductory Text for Students in Tertiary Institutions.
Abeokuta: Eagle Publishers.
5) Lawal, O.O. (2010). Library Information Practice and Education in Nigeria: Current
Trends and Issues. Calabar: University of Calabar Press

TUTORIALS
1. With the example of a named column chromatography, write a short note on
Chromatography Techniques.

2. Describe how you will obtain the crude extract of a protein?

3. Enumerate and elucidate the steps involved in purifying proteins to homogeneity.

4. Write short notes on 5 chromatographic methods that can be used in separating

proteins.

5. Described the methods you will employ in Isolation, Purification and

Characterisation of an enzyme from a micro-organism.
6. Described the methods you will employ in Isolation, an enzyme from the muscle of a
rabbit.

7. Described the methods you will employ in Isolation, an enzyme from an insect.

8. Described the methods you will employ in Isolation, an enzyme from the gut of a
snail.

9. Explain the relevance of the use of the library when writing your project.

10. Check Wikipedia and list as man search engines as possible.

NOTE: Answer these questions in an exercise book and submit immediately after
you submit your exam paper.