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Please let me clarify that it is by no means my intention to be rude to
any referees, nor to round up alternative explanations for the extra
band. The only thing I am after is a "proper" reference for the
phenomenon of his-tagged proteins producing an extra band at slightly
higher apparent molecular weight.

I just unearthed a 2007 thread from the CCP4 archives where this very
subject appears to have been discussed already
(https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind0711&L=CCP4BB&D=0&P=38996)
but unfortunately I didn't find any references to published literature
there either.

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: [log in to unmask]


On Thu, 28 Oct 2010 10:09:37 -0400, "Skrzypczak-Jankun, Ewa"
<[log in to unmask]> wrote:
> 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
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> http://golemxiv.dh.meduohio.edu/~ewa  
> 
> ******************************************************** 
> 
> -----Original Message-----
>  From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf
> Of Tim Gruene
>  Sent: Thursday, October 28, 2010 7:51 AM
>  To: [log in to unmask]
>  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: [log in to unmask]
> 
> --