Subcellular fractionation issues - MS analysis - (May/20/2014 )
Hopefully someone can help me.
I am trying to isolate membrane proteins from leukemic B-cells. We have tried 5 different methods (commercial kits, sonication, homogenisation, pressure lysis, needle homogenisation) in both hypotonic and isotonic buffers. We can bust the cells open okay (checked with trypan blue and light microscopy), we then sequentially centrifuge to isolate the nucleus, mitochondria, then a high speed spin to seperate the soluble proteins (cytoplasmic) and insoluble (membranes).
I have run the 'membrane' pellet on MS to see how clean it is, and it is FULL of histone proteins, ribonucleoproteins and bucket loads of other nuclear proteins. Interestingly, our key B-cell membrane proteins (CD19, CD40, TLR receptors, chemokine receptors etc) DO NOT show up!! Now its either an issue that the sample is swamped with so much nuclear contamination that we are not detecting it, or those components are coming down in the first 'nuclear' prep (we have dot blotted for CD40, CD19 etc and the results are inconclusive . . . . . ). I have not analysed the nuclear fraction on MS, but have done protein assays which shows lots of proteins in that pellet.
I was wondering if its possible to remove dominant proteins (like you can for albumin) but histones, RNPs etc?
Just a thought, If you pass your lysate to antibody column of the dominant protein, you can remove that protein?