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VEGF-HEK - Protocol for collecting secreted protein (Feb/16/2010 )

Hi!

I'm transfecting HEk293 cells with VEGF164, and need help for the protocol.
The protein is secredted, and I need to run western analysis on the media.

1 Should I grow the cells in serum free media after transfection, and then collect the media after 24 h? Or change to mediua with serum after transfection as usuall, and then change after some time to serum free and collect after 24 h?

2 Do I need to precipitate the proteins in the media before running western?

Thank you soo much :) :(

-Heleh-

Hi Heleh,

the following paper may be useful to you..its freely downloadable...there is a VEGF assay which talks about the various time points and use of serum free media...
"Regulation of Vascular Endothelial Growth Factor Synthesis and Release by Human Luteal Cells in Vitro"
The Journal of Clinical Endocrinology & Metabolism 91(6):23032309

hope this helps...
:(

-deva.kumari-