I have seen protocols from net which use agarose protein A/G beads. I would like to do the experiment without agarose/protein A/G beads. Can anyone please help by sharing simpler protocols for receptor phosphorylation inhibition studies using anti - VEGFR2 antibody and anti phosphotyrosine antibody?
The paper listed below contains a protocol for immunoprecipitation of VEGFR2 followed by immunoblotting with anti-phosphotyrosine antibodies; carried out following VEGF stimulation of cells. To collect the antibody bound to VEGFR2 in the immunoprecipitation it is easiest to use beads. Prior to using beads, one would use formalin fixed, heat-inactivated Staphylococcus aureus cells which express protein A and enable centrifugation to collect the antibodies. You cannot use anti-pY antibodies to inhibit phosphorylation.
Lucas, J.T., Jr., Slomiany, M.G., Salimath, B.P. and Rosenzweig, S.A. Regulation of invasive behavior by vascular endothelial growth factor is HEF1-dependent. Oncogene 29(31), 4449-4459, 2010. PMCID: 2921319
Use vanadate to inhibit phgosphatase in the lysate and pull down VEGF with a polyclonal antibody specific to the protein. Make sure the antibody does not cross-react to related proteins. Probe the immunoblot with phospho-specific antibody.
If you are first serum starving your cells, and then adding VEGF plus/minus an inhibitor, in may NOT be necessary to IP. Just Run total cell extracts, Western of phospho-Y, strip blot and reprove with anti-VEGFR. Look at this reference for a study from my lab. Oncogene (2003) 22, 3361–3370
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