I have cloned my gene of interest HBV surface gene in pRset B expression vector(prokaryotic). I have cloned 1273 bp in pRset B vector but the stop codon is present 681st nucleotide. So ideally the protein expression should stop at the stop codon. after confirming the successful expression in induced and uninduced sample on SDS PAGE I proceeded for western blot. I have used Anti-His as my primary antibody as His Tag is present at the N terminal but my western results are very confusing. The Anti-His binds more strongly at 37 kDa protein. Could it be possible that the host bacteria bl21 de3 also has his clusters? or this the case of aggregation of protein? picture attached below shows my western results. My protein size is of 28.3 kDa. Lane 1 to 6 contains the same sample expressed at same conditions. But the amount loaded in each well from 1 to 6 is in decreasing order.
Hi
In my opinion, your western blot results has no problem and appearance of protein at different molecular size than its predicted size is logical. This behavior is generally seen in structural proteins that often show unexpected electrophoretic mobilities due to differences in relative molecular mass (Mr) and Molecular Weight (MW). For example, Human Coilin protein has predicted MW of about 60 kD but its Mr is nearly 90 kD. On SDS-PAGE and western blot analysis, it shows an approximate size of 90 kD. I think your target protein (HBV) is showing similar phenomenon.
Good Luck
Usman
Hi there,
As a control for WB analysis you should run sample from strain bearing the empty plasmid to spot which bands are specific of your expressed protein and which are from the cell background. Some bacterial proteins are naturally His rich and therefore tends to interact significantly with anti-His antibodies.
I agree with Dominique Liger you should run sample from strain bearing the empty plasmid to spot which bands are specific of your expressed protein and which are from the cell background try to ran one of the interacting proteins bearing the his tag as a control. .all my best
Have you done the sequencing of the clone? If not do that. As from my experience it look that the protein coming at 37kDa and the other bands might be degradation (if lower) or nonspecific background which are often present due to his clusters in bacterial proteins as well as due to high exposure in western blot. One more thing, I would like to add that please check which site you have used for cloning, as in any vector other MCS sites and additional sequences of the vector might add up in the final mol. weight of the protein rather than only the sequence which you have cloned. Do the sequencing, most probably you will get your answer.
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