isolating phospho-proteins - After stimulating the pathway with EGF, can we still trypsinise or do we have to (Apr/06/2006 )

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We want to measure inhibition of phosphorylation of proteins in the Akt and in the MAPK pathway.
For that, we treat the cells and put EGF on them to give them a kick-in the EGFR, so the pathway is activated.

We harvested the cells bij trypinising them, and making a pellet the normal way. Then we isolated the proteins with lysisbuffer and sonification. We get a very high, good protein concentration by this method. But we got some weird results after westernblotting; proteins which should be inhibited were activated.

One of our co-workers said that the activating effect of EGF disappears really quick, and that we should not trypsinise, but just put lysis buffer on the cells immediately after the EGF.
Usually we use 200-300 µl lysis buffer on 5 million cells, but with scraping with lysis buffer in the flasks, we retrieve less cells.
Now our protein concentration is really low, to low to use.

Does Trypsinising the cells after exposure with EGF have such a big effect?

I don't know if I can help, but I will tell you how we do it...

grow cells (we use primary keratinocytes) in 6-well dishes

treat (5 ' increments -> all the rest of the steps are done very cold, very fast)

wash with ice-cold PBS

scrape

do protein extraction

if you use 6-well dishes, you should get plenty for what you need. we look at phosphorylation state as well. there is another person on this forum, I believe her name is Scarlett, that also does westerns for phosphorylation...she may have another method to help you?