I am performing whole proteome analysis. My protocol suggests boil whole cells in Guanidinium hydrochloride buffer then centrifuge followed by the usual reduction /alkylation and trypsin digestion.
In some protocols i have read a protein precipitation by acetone or chloroform methanol step after spinning of whole cell lysate and reduction /alkylation step.
Does other cellular components present in crude cell lysate when protein is not precipitated interfere with experiment in any way?
Hi Prashant,
Thank You for replying,
Yes my downstream application is MS.
I am mainly concerned about the her constituents of cell lysate effecting the number of protein identified by interfering with processing in any way.
For digestion I reduce the molarity of Guanidinium hydrochloride suitable for digestion.
But during digestion I observed a whitish precipitate forms in if use crude lysate. However there is no such precipitate when i purify/precipitate the protein first before.
I think this precipitate also correlates with the no. of peptides identified. The more it is the less the number of peptides identified.
Thanks Prashant for the insight.
This time I have given both samples i.e. with and without precipitation lets see what happens.
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