Re: [ccp4bb]: non-staining proteins??

[Laurence Pearl <laurence@icr.ac.uk>]

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Dear Neil,

One not very pleasant explanation, is that you really only have a very 
small amount of the target protein, but you get to see it in the Western 
because the immunological detection is several orders of magnitude more 
sensitive even than the silver stain. In theory if your protein doesn't 
contain tyrosines, it won't take up Coomassie terribly well, but silver 
binds to amino groups, and its a pretty rare protein that contains 
neither tyrosine nor lysine.

If you have another HA-tagged protein of reliable provenance you could 
run different amounts of that as a control lane and get an estimate of 
how much of your target protein is actually present in the band on the 
Western.

Cheers,

Laurence


Neil Isaacs wrote:

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>
>We have a detergent (DDM) solubilised membrane protein with an HA flag
>attached. Western blots with 2 different anti-HA antibodies clearly show
>the protein. However, when the blot is overlaid on the SDS PAGE gel, there
>is no match to the coomassie or silver-stained protein bands which appear
>at slightly higher MWs. The MW markers however do overlap. We can't
>explain this discrepancy (unless we assume the HA-protein is not taking up
>stain). Has anyone had a similar experience and/or able to suggest a
>reason?
>
>Thanks
>
> Neil
>
> ________________________________________________________________
>| Neil Isaacs                       Tel: +44 (0)141 330 5954     |
>| Department of Chemistry           FAX: +44 (0)141 330 4888     |
>| University of Glasgow           email: neil@chem.gla.ac.uk     |
>| Glasgow G12 8QQ                                                |
>| Scotland  U.K.                                                 |
>|________________________________________________________________|
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>  
>

-- 

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  Laurence H. Pearl

  Section of Structural Biology, Institute of Cancer Research 
  Chester Beatty Laboratories,  237 Fulham Road
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  Phone +44-207-970 6045 : Secretary +44-207-970 6046
  FAX   +44-207-970 6051 : E-Mail l.pearl@icr.ac.uk
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