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GST fusion protein problems


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#1 saad

saad

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Posted 18 May 2011 - 10:44 PM

Hi All
I have differnt truncations from the same protein with similar size between 32-35.5 kDa. I expressed them as GST fusion peotein using pGEX6p vector.
It seems to me that the fragments are not stable I could see all the soluble fractions on SDS gel about 30/31 kDa (Mark12 marker). After binding to beads and wash @4 degree on gel they seemed as three bands between ~ 20-28 kDa and two of them ran at about 36 kDa. I tried to cleave the fusion from GST then i saw to bands about 20 and 26 kDa but I can't see any bands corresponding to the size of my constructs and the two constructs that ran at ~36 kDa I saw to band matched my proteins and confirmed with MS. With Western blotting using anti-GST Ab, the tow bands are both GST even though they are differnt in size. I did pulld own with all constructs they seemed work because the results were consistent with the previous results. the question now is that what all those bands could be.

Thanks in advance for your help

Edited by saad, 18 May 2011 - 10:48 PM.





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