I tried methanol/chloroform protein precipitation method but in this method in the final step methanol and chloroform looks like single phase and It is difficult to separate methanol phase which contain protein pellet. I am looking for an alternate method.
My purpose is to separate proteins adsorbed on liposomes surface in order to quantify them using micro BCA assay. can anyone please suggest me an alternate method.
Thank you.
Try CHCL3 / n-butanol as an alternative?
Both are not miscible with H20 or each other and are easily removed with evaporation (in a fume hood of course)
Try the methanol / chloroform precipitation method by Wessel & Fluegge (see attached).
Thank you Holger Wille, previously I have done this, the problem I faced was I could not see the protein layer clearly, so there might be a chance of loosing protein while removing upper aqueous layer.
percent recovery was very less. the possible reasons for this are I might lost protein while separating aqueous layer from organic phase or I might not used a proper method to re-suspend protein pellet. I added 1ml water to protein pellet and kept it water bath at 55 deg.centig. and vortexed for 1 min and repeated the same procedure for 3 times.
If your protein concentration is very low to begin with and particularly if you protein is lipophilic, then I would use silanized tubes to reduce the amount of protein that is lost to the plastic surface. Under those circumstances the loss of protein can be quite substantial!
I used ependroff tubes (1.5ml) for this purpose.The BSA concentration I used was
60 ug/ml and lipid concentration was 1mM (100ul). I measured the absorbance of Aqueous phase and it gave very high absorbance. This is indicates that the BSA dose not get precipitated to form a layer between aqueous and chloroform phase.
Do I need to increase the speed or time of centrifugation . Could you please suggest me if there is any alternate available .
Thank you.
Hi,
you can try protein precipitation using ice cold acetone. Just add 5x volume of acetone and incubate the mixture over-night on -20°C. After fullspeed centrifugation (~30min) you can discard the acetone phase and reconstitute the protein pellet in any buffer.
Good luck
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