can i use RIPA buffer (Sigma) for isolate protein from cancer cell line for proteomic work. we have waters nanoAcquity QTof LC-MS/MS systems for analyse total proteom of cancer.
pls suggest nice protocol for my experiment
thanx in advance
RIPA works good for both LC-MS/MS and MALDI. However, there are additional steps before MS analysis.
RIPA is fine assuming that you cleanup the sample before MS analysis, ZipTip or StageTip
The RIPA buffer is not suitable for the extraction of the whole protein content of the cell. It is more suitable for the extraction of soluble cytosolic/nuclear proteins. Better go for something with more denaturing power such as 8M Urea or 6M GuCl or 2 o 4% SDS.
Here is a manuscript in which the researchers compare RIPA and Urea for protein extraction and LC-MS/MS analysis:
http://www.proteomesci.com/content/pdf/1477-5956-6-30.pdf
Thanx all for the suggestion, @ Paschalis Thomas Doulias, I have 3k filter, is it fine? i afraid the loss of protein during sample preparation. @ Ilian Atanassov, as you suggest, planning to use Urea buffer too, and the article is helpful
thank you
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