Column Chromatograhpy Column Chromatograhpy

Once the protein is expressed cells will be pelleted and the pellet will be resuspended in a suitable buffer and lysed. If present in the soluble fraction, the recombinant protein will be purified using appropriate affinity chromatographic columns.

If the protein is expressed in inclusion bodies the protein needs to be solubilized and refolded before column chromatography can be conducted (refolding).

1) Purification by affinity chromatography (column or batch)

Immobilized Tag in fusion protein
Metal (mostly Ni2+) Hexa-His
Glutathion-sepharose Glutathione S-transferase
Chitin Chitin binding sites in intein
Amylose Maltose-binding protein

 

2) Purification of tag-free proteins by

  • Cation- or anion-exchange chromatography (MonoS and MonoQ)
  • Hydrophobic interaction chromatography (Phenyl-sepharose)
  • Size exclusion chromatography
Example of step gradient Ni column purification

 

SDS-PAGE of individual fractions.

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