Dear all, I constructed plasmids with 12x His-tag at C-terminus to express the fusion proteins (64kDa). After purified by Ni2+ column, there happened to be correct bands between 55~70kDa (Coomassie blue staining), while the fusion protein in the Elution buffer could not be deteced by western bolt. I feel confused and ask anyone for help, thanks~
The only band that would appear to have the right size seems to be in the flow through fraction. This could be the strongest coomassie band but it also could be misleading. The whole elution fractions seem to indicate unspecific binding of many proteins and only a little bit of truncated target protein with his-tag. It would be helpful to see the total protein lane before the loading onnthe column. In general it's not unusual to have soluble protein with large fusion partners that are forming soluble aggregates that don't bind effectively to the column because the tag is not accessible.
You could try loading your uninduced and induced samples side by side with this flowthrough. Then, you will be sure which band is actually your protein. Also, are you using the antibody specific to your protein, or is it a His-tag antibody?
Once you are sure the band in flowthrough is your protein, then you could try purifying the protein with a new Ni-NTA column. Maybe there is some issue in the resin, which is why it's unable to capture your tagged protein, or the tag is not fully exposed, so it is unable to bind with the Ni-NTA resin!
I would agree that it seems like your protein is the strong band in flow-through. You have also some degraded peptide in the third and fourth fraction. However, those are invisible on the CB stained gel, so maybe that strong band is not your band afterall.
Anyway, apparently only degraded part of your protein binds to your column. That could indeed hint that the tag is inaccesible. so maybe try to switch it to the other side of your protein.
I would also recommend to switch to other tag, as those are usually more reliable
You've mentioned that you've done a c-terminal His tag expression. Now in case of c terminal His tags there is a problem with antibody specific binding. Majorities of the anti Poly His tag primary antibodies bind the N-terminal His tag expressing proteins better. So this looks like a problem with antibody specificity. If you mention the company name, I can recommend you something else.
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