quantitative immunoassays

Hiranya Roychowdhury hroychow at NMSU.EDU
Tue May 16 15:45:39 EST 1995


 On 15 May 1995, Fiberman wrote:

> Hello, netter, > > I wish to detect changes in the level of a specific
protein expressed in > brain tissue.  I have an antibody that will
recognize the protein.  What is > the best assay I can run to quantitate
the change in expression?  I don't need > absolute numbers, but I need an
assay that will accurately detect changes in > protein levels.  I have
tried western blots, but I feel this assay has a lot > of room for
variation.  I would appreciate any suggestions. > > -fm > > While it is
true that Western blots and subsequent ELISA may have room for variation,
the change in expression of a specific polypeptide(s) should be easily
demonstrated in a single gel, and if the technique is consistent and good,
ELISA should be fairly quantitative following Western. I wonder if you
could perform immunoprecipitation prior to running the gel, for some
proteins do not cooperate in this procedure. I would try it, nonetheless.
The best ratio of the Ab:Ag has to be figured out from the classical
Ouchterlony double diffusion test. Then the right amount of Ab has to be used
with the protein samples, keeping in mind the expected range of the 
expressed antigen. The two are incubated in 25mM phosphate buffer
(pH7.5) in microfuge tubes either O/N at 4 C or at RT for 4-6 h (these
times are empirical and varies from Ag to Ag etc.). The rxn is then spun
at the max. microfuge speed (10,000rpm) for 25 -30min. The pellet is
directly taken up in the sample buffer for PAGE. If the protein can be
labelled in vivo (or in vitro) an autorad, following the usual PAGE, will
afford nice quantitation. 


			>>>>>>>>>>>>>>>>>>>>>>>>>>>
			  Hiranya S. Roychowdhury
   			  Plant Genetic Engineering Lab.
			  Box 3GL, NM State Univ.
			  Las Cruces, NM 88003
			  Phone: (505) 646-5785
			  hroychow at nmsu.edu
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