Transcript Affinity Chromatography

Affinity Chromatography
Yongting Wang
Jan07
What is AC?
• Affinity chromatography (AC) is a
technique enabling purification of a
biomolecule with respect to biological
function or individual chemical structure.
• AC is designed to purify a particular
molecule from a mixed sample.
The resin
Affinity Ligand
Matrix
Examples of tags and ligands
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His-tag
FLAGTM peptide
Strep-tag
GST tag
Maltose binding protein fusion
Calmodulin binding protein fusion
There are situations where you don’t need a tag.
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Transition metal ion
Monoclonal antibody
Biotin
Glutathione
Amylose
Ca2+
Step 1. Loading affinity column.
Step 2. Proteins sieve through matrix of affinity beads.
Step 3. Proteins interact with affinity ligand with
some binding loosely and others tightly.
Step 4. Wash off proteins that do not bind.
Step 5. Wash off proteins that bind loosely.
Step 6. Elute proteins that bind tightly to ligand and collect
purified protein of interest.
http://www.bio.davidson.edu/Courses/genomics/method/Affinity.html
Affinity chromatography applied to recombinant proteins
Purity test
SDS-PAGE
Mass spectrometry
N-terminal sequencing, etc.
Downstream of protein purification
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Biophysical characterization
Biochemical analysis of activities
Physiological relevance
Pathological mechanisms
etc.
Example: Human lens crystallins
Human gene cloned into pET vector and expressed in E. coli.
Diagrams of crystal structure of HgD
Trp68
Fluorescence spectroscopy
Unfolded
Trp156
Native
Trp42
N-terminal domain
Trp130
C-terminal domain
Protein folding mechanisms
Electron microscopy
IR spectroscopy
• In summary, Affinity chromatography is a
method that will allow you to purify a
particular molecule from a mixed sample
so that further investigation of this
molecule could be carried out.
• Thank you for your attention.
Three groups of properties of the target molecule are used in affinity
chromatography:
1. Specific binding properties based on biological activity like:
- Enzyme active sites
- Receptor binding sites
- Antibody binding sites etc.
These are used together with the natural ligand or an analogue of it.
Sometimes the analogue has a broader specificity and can be used for
group separations.
2. Naturally occurring prosthetic groups like: polysaccharides etc.
Such properties normally allow group separations only.
3. Molecules equipped with an affinity tag like:
- Glutathione-S-Transferase (GST)
- Oligo histidine etc.
This group of properties is used almost exclusively for recombinant fusion
proteins.