Dear Sebastian, Having personally purified upwards of 500 (I lost count really) of His-tagged proteins, I can't say that I have the same awareness as you with respect to the additional band being 'very common'. Depending on the kind of expression system, size of your protein, conditions of purification, and most importantly the kind of IMAC resin you're using there can be anywhere between zero and five *extra* bands resulting from contaminants binding to the resin. In the case of E. coli expression there usually are two main contaminants (SlyD and a 61 kDa protein) but can be more than that if the ratio of your protein to resin is unfavorable. Without going through a huge PITA you can't be sure that the extra band is even related to your protein of interest - for example, you could do an in-gel digest and get peptides identified by MS however if your extra band is close enough to the main band there will always be a lingering concern that your main band is 'bleeding' into the extra band and therefore the in-gel digest results are biased towards the expected.
What sort of a concern are these reviewers trying to address? Is there some unique biological activity in the sample (like a novel enzyme activity previously unobserved for your target protein) - in which case the concerns might be justified, or is it just the level of purity with respect to structure being solved - in which case it's not much of a concern at all? Artem On Thu, Oct 28, 2010 at 6:16 AM, Sebastiaan Werten < sebastiaan.wer...@uni-greifswald.de> wrote: > Dear all, > > we have a his-tagged protein that shows a minor accompanying band in > SDS-PAGE, just above the main band. According to all other methods > available to us the material is homogeneous, the protein has the correct > mass in MALDI-TOF, epitopes are recognized, etc. etc. > > I know that the additional band is a very common artifact with > his-tagged proteins, but I was wondering if anyone is aware of a paper > that formally describes the phenomenon, as we need to appease a couple > of rather bloody-minded referees. > > Thanks very much for any suggestions, Seb. > > -- > Dr. Sebastiaan Werten > Institut für Biochemie > Universität Greifswald > Felix-Hausdorff-Str. 4 > D-17489 Greifswald > Germany > Tel: +49 38 34 86 44 61 > E-mail: sebastiaan.wer...@uni-greifswald.de >
