Internal Standard for Westerns

Basavaraju Shankarappa bsh at MED.PITT.EDU
Fri May 6 11:54:15 EST 1994


	I would directly quantitate the amount of total protein that is
going in each lane using any of the methods that are available.
I have previously done this using BCA protein assay system.  The 
sensitivity of this system is very much within the range of amount of 
protein needed for a western blot.
  
	I agree that using another antibody is going to give you a
very nice picture that can very effectively convince others.  When that
time of convincing others come, you can  resort to finding antibodies
to proteins that are the normal constituents of cell.

Raj Shankarappa
bsh at med.pitt.edu
 

> I have been looking at changes in the expression of AP-1 complexes in human
> myeloid leukemia cells treated with vitamin D and have seen some
> interesting results.  Problem is that in order to convince myself that the
> changes are real I would like to have some kind of internal standard for my
> western blots so that I know that I am looking at the same amount of
> protein on each lane.
> 
> Does anyone have any suggestions on proteins whose expression is constant
> in myeloid cells?  They need to be proteins that don't change when the
> cells differentiate or cease proliferating (which is what VD3 does to these
> cells). Obviously I need proteins that antibodies are available for.
> 
> Thanks in advance,
> SRL
> 
> -- 
> ***************************************************************
> Stephen R. Lasky, Ph.D.       Brown University/Roger Williams Medical
> Center
> e-mail: Stephen_Lasky at brown.edu         LandLine: 401-456-6572
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> A nuclear war could ruin your whole day.
> ***************************************************************
> 




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