My protein of interest is fused with MBP tag, and it does not contain tryptophan but MBP does. After factor Xa digestion, my protein was separated from MBP, and I eluted my protein while MBP is still in the MBP column.I ran SDS-PAGE with TGX stain-free gel.
Theoretically, my protein should not be visualized in Bio-Rad ChemiDoc imaging system machine because it doesn't have tryptophan. Stain-Free gels (Bio-Rad) contain trihalo compound and they interact with tryptophan residues of your protein and show fluorescent signal by UV detection.
There is no MBP contamination as it stays in the MBP column and it will only be eluted by maltose. What could be the reason?
Thank you in advance!
Can you show the gel - have you also seen what you get if you stain the gel using commassie etc? Without knowing more details about your protein and purification method it is difficult to diagnose if there is a problem.
If you know your protein doesn't contain tryptophan and therefore shouldn't show up on a stain free gel, it does make me question why you are using stain free gels to analyse your samples. It would surely make more sense to just stain the gel so you can see if your protein is definitely present or not.
I've never used factor Xa protease myself, but from my understanding it does have a big tendency to cleave at sites other than the expected one, so you could have multiple forms of protein present in your elute after the digestion, with some that is lengthened to include a tryptophan containing portion of the MBP tag.
Again, without seeing the gel and knowing your procedures, it is difficult to diagnose the problem, but I presume that you removed the Factor Xa protease (using e.g benzamidine resin) prior to preparing your samples for the gel? The protease itself will show up on a gel, so if you're getting a single band at 43 kDa (very similar in size to the MBP tag) showing up on your stain free gel it is likely the protease.
Dear Siting
the mw it corresponding to the expected one?Is it possibile that the band the you see it is due to the presence of fattXa added for the digestion?
Did you tried to stain the gel also with comassie blue to have a double check and see if you have only this band?
best regards
Manuele
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