I have expressed a protein of around 18Kda with C terminal his tag in pET21A.I have very low level of expression in pellet with protein going in pellet.I did low temperature expression to get soluble protein but find it distinguish to differentiate between uninduced ,indiced recombinant as well as pET vector control.Please advise on how to prove that my protein in soluble fraction is the recombinant.Isnt Histag one option or can i just go ahead and try with activity assays as a confirmatory test.is this called protein masking.
Sebastian's anti-His tag blot is a wise first step. Proving that your protein is "not" in the soluble fraction may be an interesting approach. Since you have written that you have proof of intact protein expression in the insoluble fraction, albeit relatively small amounts, then it is possible that the soluble expression is even less or absent by Coomassie or silver stained SDS-PAGE. The protein maybe toxic and could be degraded in the soluble fraction so the anti-His tag may be seen at much lower molecular weights or even migrate with the dye front. Is there any non-cross reacting antibody to your protein that can be used for a Western? Isolation of very lowly-expressed His-tagged proteins by Nickel NTA is difficult because of the abundance of competing host proteins that contain significant histidines in their sequence. But that could still be a clean up step for subsequent LC-MS to look for the absence of your protein.
If your enzyme has an activity that is specific enough that you can distinguish it from all the other enzymes in the cell, you can assay for it.
By the way, chromatography on nickel resin may not be sufficient to purify a poorly-expressed His-tagged protein in a single step. You may need to combine multiple chromatography methods if you can't improve expression.
@Adam:Yes I do have an activity assay and I have shown activity with 2 batches-now I need to do the same with controls of uninduced as well as vector induced and uninduced.When I bound and eluted thee controls to Nickel NTA I see a huge amount of background but I guess if i show activity of my clone "inspite "of the background I will be fine along with other purified proteins known to have no activity against the substrate .
And yes maybe I need to do ion exchange either or after His tag chromatograohy.When I did His tag chromatography after denaturing His tag I got my expected size protein bound and eluted so revisiting that in terms of trying refolding may not be a bad idea.
And yes @ Sebastian checking with His tag antibody at all steps is a good idea which I am already exploring.
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