I am using methanol/ choloform mixture for protein extraction from other interferring biomolecules, e.g: lipid, other peptides etc. Now I use 200 ul of protein sample with 3: 0.75: 2 volumes of methanol: CHCl3: H2O with respect to the protein volume (as per thermofischer Click protein reaction buffer protocol). I am getting three layers, bottom one for organic and the upper one for the aqueous layer. After discarding the aq layer, I put 2.25 times volume of the protein sample for a final wash, vortex , centrifuge and then discard all the supernatant, but when I suspend the pellet in PBS and try to find out the protein concentration by Nanaodrop, there I get very small quantity.
Any one please help
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