Thanks for your response. (Forgot to check back).
What I do not understand is that the commercially derived protein runs at the expected sizes even under non reducing conditions.
What can I do to fix this?
Whenever I add DTT it has no effect that I can see on the SDS Page. The band at 32kDa stays the same. VEGF has 7 disulfides, two of which form bridges between the monomers to form the dimer. It is a cysteine knot motiff if that helps. I add 5ul of 1.25M DTT to a solution of 35ul containing my protein and the loading buffer.
I did a proliferation assay and my protein is active (nearly the same as "standard" vegf) and is detected by ELISA, but Im having a hard time convincing my PI (and myself) that the wrong size is ok.
I just did another round of purification using a slightly different tag on the VEGF (it is a fibrin binding domain with 7 amino acids). So this raises the MW by less than 1kDa. However I am seeing the same thing.
I do have one question that may help me but is not directly related. I am using a pET-28a vector and when I use clone manager it has to transcribe and translate in "complementary". Does this have any effect on protein expression?
Again any help on forming this dimer correctly is greatly appreciated. (I have tried refolding from IB's with multiple methods with no results unfortunately).
Randy SmithMember Since 02 Nov 2012
Offline Last Active Nov 12 2012 10:15 AM
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protein expression and purification, vascular engineering
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09 Nov 2012 - 03:30