Western/Signaling Studies - How to get rid of background phosphorylation? - Western/Signaling Studies - How to get rid of background phosphorylation? (Feb/12/2006 )

Dear all,

I have been trying to do signaling studies on murine peritoneal macrophages, looking at the effect of various stimulants on the phosphorylation of key signaling molecules.

Basically, I stimulate the cells with various agonists and obtain lysates from the cells. Then I run the lysates on an SDS-PAGE, transfer onto PVDF membrane, and probe with antibodies such as anti-phosphotyrosine, anti- phospho IkB alpha etc.

The problem is that I have been getting very high background phosphorylation in the unstimulated controls, which probably masks any effect the stimulants have on phosphorylation. I have tried starving the cells in serum free medium (DMEM, 2% BSA, 20mM HEPES, Pen/Strep) for 4hr and 24hr, but this did not seem to have any effect at all. How do I get rid of the background phosphorylation?

Would be grateful for any comments/advice/suggestions.

i had the same problem many many times....i starved the cells longer....go up to 36 or 48 hours(compatible with their viability, of course) and i also tried different cell lines.

Cheers for the reply.

I have actually thought about starving my cells for longer, but the thing is I am working with primary cells from mice, which are less viable than cell lines (e.g. my cells are ~60-65% viable after starvation for 24 hrs) What starvation medium did you use? Also, what cells/cell lines did you work with?

Another thing is I am working with macrophages, which are very "sticky" (they bascially adhere to any surface). I have read that signaling through integrins can lead to protein phosphorylation, so I am trying to think of some ways to incubate the cells in starvation medium without letting them stick. Any suggestions?

i worked with endothelial cells; i just went down to 0.05%-0.1% FBS (or no serum at all with hybridoma cells which are more resistant) DMEM 1% antibiotics/antimicotic and 36-48hr starvation; your cell line would not survive this conditions. sorry, i dont have experience with macrophages but i guess you could grow them in suspension. also check which medium other people use for macrophages....maybe is not DMEM or you have to adjust conditions (Ca++ or other ions/aminoacids contained in the medium) to grow them better.