PBS for releasing anchored cells, what kind? - (Dec/04/2008 )

I need to try a new method of getting my cells to release during lysing. The RIPA buffer I am using is not releasing the cells and I have to really scrape hard, and that still leaves cells. I want to release the cells using PBS, but I'm not sure what kind to use. I don't want to chance it with Trypsin because I can't risk my protein getting cut. One person told me to use PBS with Magnesium chloride and Calcium chloride as that releases the cells safetly, and I have read a post here that said that Calcium chloride helps to prevent cells from releasing. So which is it? I need all the little buggers to release.

I don't know why anyone would recommend Ca or Mg, as integrins require divalent cations for adhesion. That said, you might consider a buffer that chelates the divalents. Depending on how you're going to quantify your protein you could try EDTA or citrate buffers - just beware that BCA won't work with these buffers, because they'll sequester the Cu in the color reagent.