Problems with protein expression

Qingwu Shen via methods%40net.bio.net (by shenq from purdue.edu)
Fri Sep 19 09:42:32 EST 2008


Take and others,

Here is more detailed information. The vector I'm using is pET-3d, ampicillin 
resistant gene,T7 promoter. After I harvested the cells, I sonicated them in 25 
mM Tris/pH 7.5, 5 mM EDTA, 1 mM DTT, and 8 M urea. Urea was used to dissolve the 
protein I was trying to express (TnI). After centrifuge (32,000 X g) to remove 
cell debris, I mixed the supernatant with SDS-PAGE loading buffer (containing 8 
M urea) and the load samples  on to gel. I think there should be no problem 
about protein extraction because I used the same protocol to extract both human 
cardiac TnI and wild type rabbit skeletal TnI. It works for both. Only after I 
mutated the rabbit skeletal TnI using PCR, I got no protein I wanted. Somebody 
suggests to re-clone the gene to another vector, do you guy think this will 
really works? What really confuses is that the wild type can be expressed, but 
the mutant can not. Anybody can explain this? Your help are really highly 
appreciated.

Best regards,

Shen

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