Hi Sebastian, Under the assumption that the SDS in your assay does not completely unfold the protein during electrophoresis (chemical impurity can be excluded because of MS experiments, right?), how about adding some urea additionally to the SDS-PAGE (or changing SDS concentration)?
GL Jan --- Skrzypczak-Jankun, Ewa <ewa.skrzypczak-jan...@utoledo.edu> schrieb am Do, 28.10.2010: Von: Skrzypczak-Jankun, Ewa <ewa.skrzypczak-jan...@utoledo.edu> Betreff: Re: [ccp4bb] Additional band on gel due to his-tag: any references? An: CCP4BB@JISCMAIL.AC.UK Datum: Donnerstag, 28. Oktober, 2010 16:09 Uhr Additional band on a gel might not be caused by his-tag. It is often a result of different conformation/molecular shape and so the molecule travels with different speed in the gel. We may wish for a homogenous sample (chemically and structurally) but this is seldom true See example: Int J Mol Med 2009, 23(1), 57 Jankun et al, VLHL plasminogen activator inhibitor spontaneously reactivates from the latent to active form. I fully agree with Tim - making rude comments about reviewers (or anybody else) is not going to help you. Questioning an extra band is a legitimate remark that you should address. Good luck - Ewa ******************************************************** Dr Ewa Skrzypczak-Jankun Associate Professor University of Toledo Office: Dowling Hall r.2257 Health Science Campus Phone: 419-383-5414 Urology Department Mail Stop #1091 Fax: 419-383-3785 3000 Arlington Ave. e-mail: ewa.skrzypczak-jan...@utoledo.edu Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa ******************************************************** -----Original Message----- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tim Gruene Sent: Thursday, October 28, 2010 7:51 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Additional band on gel due to his-tag: any references? Dear Sebastiaan, isn't it the editor rather than the referees whom you have to convince? And if the editor does not even understand how SDS-PAGE works and still considers this a reason not to publish your article against your own expertise, maybe it is worth changing the journal. Finally, since referees know the names of the authors of the articles they are refereeing, you may need a lot more than a couple of references to appease them after you called them "bloody-minded" in a public email forum. My two cents, Tim On Thu, Oct 28, 2010 at 01:16:59PM +0200, Sebastiaan Werten 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 -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A
