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Synthetic phosphopeptide immunogens yield activation-specific antibodies to the c-erbB-2 receptor
R J Epstein1, B J Druker, T M Roberts
1Division of Cell and Molecular Biology, Dana-Farber Cancer Institute, Boston, MA.
Abstract:
We inoculated rabbits with synthetic phosphopeptides, duplicating a major autophosphorylation site of the c-erbB-2 protooncogene product. The rabbits produced antisera that, after reverse immunoaffinity purification, selectively recognize the erbB-2 protein in its enzymatically active configuration. These anti-phosphopeptide antisera identify a subset of erbB-2-positive human cell lines wherein the protein is constitutively active as a tyrosine kinase. Synthetic phosphopeptides incorporating informative protein phosphorylation sites may prove useful for generating antibodies that indicate the activation state of additional tyrosine kinases and perhaps other proteins phosphorylated on serine and threonine residues.
Insights
Researchers developed antibodies targeting the active form of the c-erbB-2 protein by using synthetic phosphopeptides. These antibodies can identify specific human cell lines with constitutively active tyrosine kinase activity, aiding in cancer research.
Area of Science:
- Molecular Biology
- Oncology
- Immunology
Background:
- The c-erbB-2 protooncogene product is a key protein in cell signaling.
- Understanding the activation state of c-erbB-2 is crucial for cancer research.
- Tyrosine kinase activity is often dysregulated in cancer.
Purpose of the Study:
- To generate antibodies that specifically recognize the active, phosphorylated form of the c-erbB-2 protein.
- To identify human cell lines with constitutively active c-erbB-2 tyrosine kinase activity.
Main Methods:
- Inoculation of rabbits with synthetic phosphopeptides mimicking c-erbB-2 autophosphorylation sites.
- Production and reverse immunoaffinity purification of rabbit antisera.
- Testing antisera against human cell lines to detect active c-erbB-2.
Main Results:
- Generated antisera selectively recognized the enzymatically active configuration of the erbB-2 protein.
- Identified a subset of erbB-2-positive human cell lines with constitutively active tyrosine kinase activity.
Conclusions:
- Synthetic phosphopeptides are effective tools for generating antibodies against specific protein phosphorylation states.
- The developed anti-phosphopeptide antibodies can serve as biomarkers for active c-erbB-2.
- This approach may be applicable for developing antibodies for other activated kinases and phosphorylated proteins.