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Binding of proteins, including pp60src, to activated CH-sepharose 4B
N Pavloff1, J M Biquard, M Mariller
1Groupe 8 du C.N.R.S., Institut Gustave-Roussy, Villejuif, France.
Molecular Biology Reports
|January 1, 1987
Summary
Researchers identified the Rous Sarcoma Virus (RSV) transforming protein, pp60src, in chick embryo cells using affinity chromatography. This protein binds to Sepharose beads and can be eluted with dimethylsulfoxide.
Area of Science:
- Biochemistry
- Molecular Biology
- Virology
Background:
- Affinity chromatography is a powerful technique for protein purification.
- Chick embryo cells are a common model system for studying viral oncogenes.
Purpose of the Study:
- To identify and characterize polypeptides bound by activated CH-Sepharose 4B and protein A Sepharose CL-4B from chick embryo cells.
- To determine the identity of major bound polypeptides, including potential viral transforming proteins.
Main Methods:
- Affinity chromatography using activated CH-Sepharose 4B and protein A Sepharose CL-4B.
- Analysis of bound polypeptides by molecular mass.
- Immunoblotting to identify specific proteins.
- Elution studies using various chemical agents.
Main Results:
- Sepharose matrices selectively bound polypeptides of 57-60 kDa and 47-49 kDa from chick embryo cells.
- These bound polypeptides were resistant to elution by detergents, high salt, and reducing agents.
- Immunoblotting identified one 57-60 kDa polypeptide as the Rous Sarcoma Virus (RSV) transforming protein, pp60src.
- pp60src was eluted by 60% dimethylsulfoxide but not by SDS, beta-mercaptoethanol, NaCl, or Tween 20.
Conclusions:
- Activated CH-Sepharose 4B and protein A Sepharose CL-4B can effectively isolate specific polypeptides from cell lysates.
- The RSV transforming protein pp60src can be purified using these affinity matrices.
- Dimethylsulfoxide is an effective eluent for pp60src from the immunoabsorbent, suggesting specific binding interactions.