286676242-Affinity-Chromatography-ppt.pdf

introduction

1. Affinity Chromatography

2. What is Affinity Chromatography???
Affinity Chromatography is a method from
which we can separate biochemical mixtures.
The separation is based on highly specific biological interaction.
Examples:-Antigen and Antibody,
Enzyme and Subtract
•By the use of Affinity Chromatography we can separate proteins
on the basis of reversible interaction between protein and
specific ligand.
•This technique offers high selectivity,
high resolution and
high capacity for the protein.
•Affinity Chromatography is unique purification technology

3. Common terms in Affinity
Chromatography
•Matrix: for ligand attachment. Matrix should be
chemically and physically inert.
•Spacer arm: used to improve binding between
ligand and target molecule by overcoming any
effects of steric hindrance.
•Ligand: molecule that binds reversibly to a
specific target molecule or group of target
molecules.
•Binding: buffer conditions are optimized to
ensure that the target molecules interact
effectively with the ligand and are retained by the
affinity medium as all other molecules wash
through the column.

4. •Elution: buffer conditions are changed to reverse
(weaken) the interaction between the target
molecules and the ligand so that the target
molecules can be eluted from the column
•Wash: buffers that wash unbound substances
from the column without eluting the target
molecules or that re-equilibrate the column back
to the starting conditions
•Ligand coupling: covalent attachment of a ligand
to a suitable pre-activated matrix to create an
affinity medium
•Pre-activated matrices: matrices which have been
chemically modified to facilitate the coupling of
specific types of ligand.

5. Principle of Affinity Chromatography
•Inject a sample into an initially equilibrated
affinity chromatography column
•Only the substances with affinity for the ligand
will retained in the column.
•Other substances with no affinity for the
ligand will eluted from the column.
•The substances retained in the column can be
eluted from the column by changing pH or salt
or organic solvent.

7. Method of Affinity Chromatography
•Binding of the selected ligand to the matrix requires that a covalent
bond be formed between the two which is facilitated by
derivitization of the sugar residues' hydroxyl groups.
•It is important to realize that the substrate might not be able to
reach the ligand active site if it is hidden deep within the ligand.
Therefore, most ligands are attached first to spacer arms which are
then bonded to the matrix.
•The ligand-matrix gel is then loaded into an elution column.
•Once the column has been prepared, the mixture containing isolate
is poured into the elution column.
•Once in the column, gravity pulls the solution through the gel,
because most of the proteins do not bind to the ligand-matrix
complex.
•However, when the ligand's recognized substrate passes through
the gel, it binds to the ligand-matrix complex, halting its passage
through the gel.
•Some of the impurities flow through the gel due to gravity, but
most remain, unbound, in the gel column.

8. •In order to remove these unbound impurities, awash of
extreme pH, salt concentration, or temperature is run through
the gel.
•It is important to use a strong wash so that all the impurities
are removed, but it is also just as crucial that the wash be not
so strong that it removes the bound isolates.
•Once the impurities are washed-out, the only remaining part
of the protein mixture should be the desired isolates.
•Finally to collect your favorite isolate, which is still bound to
the ligand-matrix in the gel, a stronger second wash is run
through the column.
•This second wash relies on the reversible binding properties
of the ligand, which allows the bound protein to dissociate
from its ligand in the presence of this stronger wash.
•The protein is then free to run through the gel and be
collected.

10. The Chromatogram
•Affinity chromatography is not just limited to isolating one
protein; given a sample of similar proteins with similar binding
affinities, a chromatogram can be generated.
•A chromatogram is a plot of absorbance vs. time for the
elution of proteins in affinity chromatography.
•Four things may be learned from a chromatogram:
1)The level of complexity of the sample (indicated by the
number of peaks)
2)Qualitative information about the sample composition (by
comparing peak positions with known standards)
3)Quantitative information of the relative component
concentrations (by comparing peak areas)
4)Total column performance (by comparing with known
standards).

11. Chromatogram

12. Applications of Affinity
Chromatography
Affinity chromatography can be used to:
•Purify and concentrate a substance from a
mixture into a buffering solution
•Reduce the amount of a substance in a
mixture
•Recognize what biological compounds bind to
a particular substance
•Purify and concentrate an enzyme solution.

13. Case Studies.
•Determination of binding constants
byaffinity chromatography
•In the use ofaffinity chromatographyto characterize biospecific
interactions in terms of reaction stoichiometry and equilibrium constant.
In that regard, the biospecificity incorporated into the design of the
experiment ensures applicability of the method regardless of the sizes of
the reacting solutes.
•By the adoption of different experimental strategies
(columnchromatography, simple partition equilibrium, solid-phase
immunoassay and biosensor technology protocols) quantitativeaffinity
chromatographycan be used to characterize interactions governed by an
extremely broad range of bindingaffinities.
•The link between ligand-binding studies and quantitativeaffinity
chromatographyis illustrated by means of partition equilibrium studies
ofglycolytic enzymeinteractions with an exercise which emphasizes that
the same theoretical expressions apply to naturally occurring examples
ofaffinity chromatographyin the cellular environment.
(Ref: Journal of Chromatography AVolume 1037, Issues 1-2, 28 May 2004, Pages 351-367)

14. •Experimental studies onaffinity
chromatographyin an electric field
•A multi compartment electrolyzer, which has been used for preparative
electrophoresis [Z. Liu, Z. Huang, J.-Y. Cong, et al., Sep. Sci. Technol. 31
(1996) 427], is applied for carrying outaffinity chromatographyin an
alternating electric field.
•The effect of electric field strength on the adsorption and desorption
characteristics is experimentally examined with human serum albumin
and Blue Sepharose Fast Flow as a model system.