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6X His problem w/ pET45b vector
Posted by: Syn (IP Hidden, New member, 1)
Date: October 7, 2005 08:31PM

HELP!

I've got a synthetic construct (codon optimized gene) in pET45b KpnI site with an N-terminal 6xHis tag. Sequence looks good. When I check induction by coomassie, there is clearly an induction-specific band at the correct molecular weight that is absent in the negative control. This band is also found when inducing the native sequence construct (also in the pET45b KpnI site). The gene contains a GPGP linker between the histag and the coding sequence.

Here's the problem: when we try a western with a 6x His antibody, we get no response except from the positive control lane (well, once we got a *faint* band at the correct MW). There isn't any out-of-phase coding frames that would generate an expected protein.

So what is going on?!?



Edited 1 times. Last edit at 10/07/05 08:32PM by Syn.

 

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