I want to compare the protein expression level of different bacterial transcriptional regulators in E. coli. Are the GFP or other fluorescent protein genes suitable for determining protein expression? I have tried western blot, but the protein bands are too weak when using His tag. any suggestions?
You could use any indicator gene like GFP, or you could directly measure the RNA transcript being made through RT-qPCR which might be more accurate.
Michael J. Benedik Dear Michael J. Benedik, thanks for your kind reply. I have tried RT-qPCR and lacZ reporter gene to measure the transcriptional changes, but it has no difference at mRNA level to explain the phenotype change. So I guess there are Post-transcriptional regulation or translation regulation control. Any suggestion? Thanks a lot :)
RT-qPCuR should be the best way to truly measure transcriptional changes. But of course you could have some form of translational control or RNA processing, however the lacZ should have shown that. Lastly it could just be a matter of protein turnover, some specific regulated process that degrades or processes the protein. You might try a different tag than His tag, I don't think the signals with His tag are as good as some of the other tags for western blotting, so another tag might get you enough signal to detect the differences.
Lastly consider that the phenotypic changes may not be at the level of the protein quantity, but rather at its activity.
I agree with your idea trying another tag for western blotting. As for the activity of regulator, I have also used EMSA and SPR methods to compare the affinity between DNA and my regulators. However the results could not answer my phenotype difference question. Thanks again for your kind suggestion! I will try flag tag or other tag! :)
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