I've faced a weird problem. My target DNA methyltransferase was cloned into pTrcHisA expressionvector. The expression was carried by both IPTG and autoinduction system. After induction I've tried to purify plamid and protein extract independently. Plasmid purified by alkaline lysis and protein was extracted by sonication. I checked plasmid that was methylated, but there was nothing in western blotting. I am sure that everything for western is okay.
The E. coli you are using for expression do not naturally methylate DNA at the same site your methyltransferase does, I assume? What is your assay for methylation?
After sonication did you spin down the insoluble material and only run the soluble fraction, or did you run total lysate? Do you know if your protein is soluble in the conditions used to lysed the E. coli? How sensitive is your anti-methyltransferase Ab in western?
Yeah I ran both of soluble and insoluble. And I used anti-his antibody for western blotting. My target protein is soluble.
OK, that's a start.
Though you said everything was fine with your western blot, and by this I assume you mean that your positive control His-tagged protein gave great signal on the same blot that was negative for your protein of interest, anti-His antibodies are notoriously bad in western blot (and any other sort of application IMHO). Was the anti-His Ab directly labeled with peroxidase or did you use a peroxidase-labeled secondary against the anti-His? I am getting at whether you used conditions that were highly sensitive in order to detect a possibly low expressing protein using a weak antibody.
Also, you didn't answer my question about the methylated DNA.
My protein is a CpG specific methyltransferase . I used hpaii and mspi to digest plasmid. And western blotting I used secondary antibody with HRP conjugation for detection.
With a CpG-specific MT and those REs you certainly should get a specific answer about methylation activity. Using a secondary-HRP Ab is the better way to go, so that's good.
Could be that the His tag is not on the protein. Has the vector sequence and recombinant protein be validated before?
Could be a combination of the anti-His antibody being weak and the protein expression being low. The protein may be expressed at a low level - enough to be functional, but not enough to be detected by your anti-His antibody conditions. How much His-tagged control protein did you run in your western blot to confirm that the assay worked?
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