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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. .

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affinity chromatography

Affinity Chromatography

Yongting Wang

Jan07

what is ac
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.
slide3

Affinity Ligand

Matrix

The resin

examples of tags and ligands
His-tag

FLAGTM peptide

Strep-tag

GST tag

Maltose binding protein fusion

Calmodulin binding protein fusion

Transition metal ion

Monoclonal antibody

Biotin

Glutathione

Amylose

Ca2+

Examples of tags and ligands

There are situations where you don’t need a tag.

slide10

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

purity test
Purity test

SDS-PAGE

Mass spectrometry

N-terminal sequencing, etc.

downstream of protein purification
Downstream of protein purification
  • Biophysical characterization
  • Biochemical analysis of activities
  • Physiological relevance
  • Pathological mechanisms
  • etc.
example human lens crystallins

Trp68

Trp156

Trp42

Trp130

N-terminal domain

C-terminal domain

Example: Human lens crystallins

Human gene cloned into pET vector and expressed in E. coli.

Fluorescence spectroscopy

Diagrams of crystal structure of HgD

Unfolded

Native

slide16

IR spectroscopy

Electron microscopy

slide17
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.