Affinity Purification Affinity Purification

Affinity purification is a powerful method to purify the protein of interest and to study protein-ligand and protein-protein interactions.  This method is based on the reversible interaction between the protein of interest and a specific ligand.  This method can be used to purify the protein of interest and together with its binding partner(s) from the cell extract.

Affinity chromatography can be carried out in a variety of ways: 

A. If a protein binds to a specific ligand, this ligand can then be conjugated onto beads to be used to extract the protein of interest.

B. One can also link an antibody directed against the protein of interest onto beads and purify the protein of interest and potential binding partner(s) by immunoaffinity chromatography (immunoprecipitation).

C. The protein of interest can be immobilized onto beads and binding partner(s) of this protein can be extracted from the cell lysate.

The Protein Core can produce the recombinant protein needed for (C) and conjugate either the protein or antibody onto Sepharose beads.  The Core will also perform either small-scale or large-scale affinity purification of proteins of interest for investigators.  The Core can carry out subsequent SDS PAGE and western blotting analyses.  If the interest of investigators lies in determining potential novel interacting partners to the protein of interest, the protein bands from affinity chromatography can be excised from the gel and identified by mass spectrometry via the MUSC Proteomics facility.

 

Purification of potential cellular binding partners by kallistatin affinity chromatography.  Rat VSMC lysate was subjected to affinity purification with a kallistatin affinity column.  The eluant was analyzed by SDS polyacrylamide gel and stained with Coomassie blue.  Potential kallistatin binding partners are marked by *.

Contacts Contacts

Director: Yi-Te Hsu, Ph.D. Associate Professor
hsuy@musc.edu
Tel: 843-792-0849
Co-Director: Erika E. Büllesbach, Ph.D. Associate Professor
bullesee@musc.edu Tel: 843-792-9926
Research Specialist II: Mr. Dzmitry Fedarovich
fedarov@musc.edu Tel: 843-792-6529