Agarose Gel Electrophoresis Estimate the size of molecules Agarose in AGE Gel Loading Buffer Nucleic Acid Stain Factor affecting mobility of DNA Factor affecting Resolution Smearing

1. PRESENTED BY:
PRASHANT V C
DEPT OF ZOOLOGY
GUK

2.  First developed by Pedro Cuatrecasas and
Meir Wilcheck in 1968.
 Used to study enzymes and other proteins.
 Relies on affinity of various biochemical
molecules with specificity.

3.  A method of seperating mixtures based on
highly specific interations/affinity between
the an immobilised ligand and target
molecule.
 Ex. Antibody/antigen, enzyme/substrate, and
enzyme/inhibitor interactions.

4.  Based on 3 aspects :
1. Matrix : for ligand
attachment.
2. Spacer arms : create
space between
ligand and matrix. If
they are close to
each other, then due
to steric hindrance,
the target molecules
won’t be able to bind
to the ligand.
3. Ligands : which has
an affinity for target
molecule. (reversible)

5.  Provides attachment to affinity ligands.
 Amino , hydroxyl , carbonyl, etc. groups serve
as ligand binding sites.
 Should be stable enough in various
conditions like pH, salt concentrations etc.
 Should be porous ( surface area).
 Made of agarose, polyacrylamide, cellulose,
silica etc.

6.  Selection of ligand influenced by 2 factors:
I. Ligand must exhibit specific and reversible
binding affinity for target.
II. Must have chemically modifiable groups that
allow it to attach to matrix.
• Ligands are attached to matrix by covalent
bonds using functional groups located in
matrix such as:
 Amine
 Carbonyl
 hydroxyl

7. Commonly used ligands Affinity
Concanavalin A (lectin) Sugars, glycoproteins
Wheat germ agglutinin (lectin) N-acetylglucosamine
Avidin (protein) Biotin containing enzymes
Proteins A and G Immunoglobulin IgG
Poly (A) RNA containing poly (U) sequences
Antibody
(Immuno Affinity Chromatography)
Antigen
Histones DNA
Acriflavin (antiseptic) Nucleotides
Lysine rRNA
Metal ions (Cu+2 , Ni+2 , Zn+2)
(IMAC- Immobilised Metal ion
Affinity Chromatography)
Histidine containing proteins
Hormone Receptor/ binding protein

8.  Sample injected into column.
 Wash buffer – non target molecules elute off.
 Ligand/target complex will remain in column after wash buffer elutes
off non target molecules.
 Elution buffer – disrupts interaction between target molecules and
stationary phase/ligand. so target molecules are obtained.

10.  Extremely high specificity.
 High degrees of purity can be obtained.
 Reduce the amount of substance in a
mixture.
 Used in Genetic Engineering- Nucleic acid
purification.
 Antibody purification from blood serum.
 To observe which biological compound bind
to a particular substance.

11.  Expensive ligands
 Leakage of ligands
 Degradation of solid support
 Non specific adsorption

12.  https://en.wikipedia.org/wiki/Affinity_chromatography
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469/
 Pictures available at:
• https://www.shodex.com/en/da1/09/0201.html
 https://www.quora.com/What-is-affinity-chromatography
 https://www.slideshare.net/rajpalchoudharyjat/affinity-
chromatography