Dear all,
I performed a 10 % TCA-acetone precipitation on a protein sample with high salt concentration (800 mM NaCl). After sample preparation, this sample was loaded on a 2D SDS-PAGE gel. Protein separation in first dimension (isoelectric focusing) was of low quality. I guess this was due to ionic impurities. Probably, too many salts were still present in the sample which disturbed the isoelectric fosucing process. Would it be possible that salts co-precipitate with proteins when performing a 10 % TCA-acetone precipitation? Has anyone experience with this or does anyone experience the same problem?
Best wishes,
Valérie
Hi Robert,
I wash the pellet 3 times with ice-cold acetone, but then I still have the IEF problem. I once performed a dialysis (1 night) of the sample after it was TCA-acetone precipitated to remove the remaining salts (and TCA) and then the IEF focusing was succesful. So that's why I thought that salts co-precipitate with the proteins during the TCA-acetone precipitation. If acetone is responsible for the co-precipitation of salts, washing the pellet with acetone will be 'useless' to improve the IEF.
Many thanks for your interest and your help.
Kind regards,
Valérie
Under usual or "normal" salt concentration up to some 150 mM salts TCA precipitation works fine, but you have very high molarity of salt and that is why you have a problem. You have answers given for that and just do what was suggested. Good luck.
You could try the methanol/chloroform method:
@ARTICLE{Wes-84,
title = {A Method for the quantitative recovery of protein in the presence of detergents and lipids},
author = {D. Wessel and U.I. Flügge},
journal = {Anal. Biochem.},
year = {1984},
pages = {141-143},
volume = {138},
abstract = {chloroform/methanol precipitation},
doi = {10.1016/0003-2697(84)90782-6},
language = {engl},
}
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