pMAL problem

gheeya at gmail.com gheeya at gmail.com
Wed Sep 6 15:55:17 EST 2006


Hello everyone,
I have been working on pMAL system from New England Biolabs. I have
cloned mutant of protein of interest in MCS of pMAL. After induction, I
am trying to purify the protein. I am performing Ammonium sulfate cuts,
amylose column, Q column, and SP column. After SP column, the eluted
sample is relatively clean ( there are some truncation fragments
though). The protein of interest does not stay in solution after
cleaving MBP.

The fusion protein is in buffer: 10% glycerol, 0.01% triton, 20 mM Tris
7.5, 100 mM NaCl.

I have tried changing buffer: I changed Tris with MOPS, CHES, HEPES.

I have tried increasing glycerol from 10-50%

I have tried changing triton concentration.

Adding sarcosyl does not seem to help either.

None of the above have worked so far.

Any suggesting to keep the protein of interest in solution. Please let
me know. Thanks.



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