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Immunoaffinity purification of tyrosine-phosphorylated cellular proteins
S B Kanner1, A B Reynolds, J T Parsons
1Department of Microbiology, University of Virginia School of Medicine, Charlottesville 22908.
Journal of Immunological Methods
|June 2, 1989
Summary
Researchers purified tyrosine phosphoproteins, including pp60src, from cells using anti-phosphotyrosine antibodies. This method efficiently isolates key proteins involved in viral oncogene signaling and cellular transformation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Viral oncogenes encode tyrosine kinases that drive cell transformation.
- Cellular transformation is associated with increased tyrosine phosphorylation of proteins.
Purpose of the Study:
- To identify cellular targets of oncogenic tyrosine kinases.
- To develop a method for purifying tyrosine phosphoproteins.
Main Methods:
- Immunoaffinity chromatography using antibodies to phosphotyrosine.
- Purification from rat-1 cells and chicken embryo cells expressing src oncogene variants.
- Elution of bound proteins using hapten.
Main Results:
- Purified 6-10 highly pure phosphoproteins, including pp60src.
- Recovered proteins represented ~0.03% of total cellular proteins.
- All purified proteins contained phosphotyrosine and were also phosphorylated on serine and threonine.
Conclusions:
- Developed a large-scale, single-step purification method for phosphotyrosine-containing proteins.
- The method yields proteins pure enough for immunization and further characterization.
- Identified key phosphoproteins involved in src oncogene-mediated cellular processes.