Control mAb needed.

mills_j at mscf.med.upenn.edu mills_j at mscf.med.upenn.edu
Tue Feb 23 01:26:34 EST 1993


 rpgrant at molbiol.ox.ac.uk writes:
> Hello again, bionauts.
> 	I am doing Westerns of total (cultured) cell protein in an effort to 
>find out what happens when I transfect cells with my favourite DNAs.  I need a 
> loading control (obviously), but I've looked through three catalogues and 
> found nothing suitable and it's Friday and I'm depressed......
> I am looking for a * mouse * monoclonal (IgG) (so that I can use the same 
> secondary HRP-ab) against a constituitively expressed protein in the order 
> of 50 - 100 kDa.  The protein must be intracellular and NOT associated with 
> the cytoskeleton. The nearest I've found is abnti-PTPase.
> BTW, I'm working with mouse and dog fibroblasts.  
> HELP!
> -- 
> Richard P. Grant                        rpgrant at molbiol.ox.ac.uk

    I wish I had your answer, but, unfortunately, I looked into this whole
control-Ab issue a lot more than it sounds as though you've done, and I 
couldn't find anything too helpful.  I wanted a mouse monoclonal too, against
something constitutive in rat neuronal cells (I would have taken 
anti-cytoskeletal).  Trouble is, if you go commercial, everything is so 
expensive that use as a control is pretty much precluded.  Plus, everyone I 
talked to seemed to think I was crazy to want a loading control on a western: 
"People only do loading controls on northerns", they would say.  Yet I never 
got a satisfactory answer as to why one can't want a loading control for 
proteins as well.
    I _did_ do a decent amount of catalog browsing and phone-calling.  I'm 
afraid the only things I found that came close to acceptable were 
anti-cytoskeletal.  Another thing to try, though, now that I think about it, is 
Ponceau S reversible staining your blot to check overall loading and transfer 
first, before immunostaining.  I haven't actually tried this yet, but it sounds 
good on paper.

-- 
                                  JCM                      
             |---------------------------------------------|
             |--Mills_J at a1.mscf.upenn.edu (Jason Mills)----|
             |"Why would anyone want both an MD and a PhD?"|
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