Dr. Hiranya S. Roychowdhury
hroychow at nmsu.edu
Thu Dec 18 17:14:28 EST 2003
I am stumped too! Here is an idea which may not be too outlandish. Your
experimental protocol permitting, you could label the protein in vivo (35S) for
your different mitochobdrial samples. Then do an autoradiography of the gel.
The autoradiogram can be used to show th quantitative variation across the gel
for your protein of interest (this is assuming that you can actually detect the
polypeptide on a gel. BTW, around 20kDa is not too small. I am not sure how
you could actually quantify any protein without some sort of stoichiometry.
I'll think some more.
Quoting "Jayakumar, R" <R.Jayakumar at RoswellPark.org>:
> I have a protein quantification problem. I have several crude
> mitochondrial protein extracts, in which I have to quantify a particular
> protein of interest and show the extent of variation in quantity of the
> particular protein in each of the extract. Though the protein is an enzyme,
> for several reasons, I cannot do an enzyme assay for quantification. I have
> polyclonal antibodies against the protein of interest but I cannot do
> immunoprecipitation since I have very little of the antibody left and it is
> impossible to raise the polyclonal antibody again (that is a long story).
> I have so far tried using western blots for quantification, but found it
> very unsatisfactory, since results are not very repetetive, and the slight
> variations in protein binding across the blot makes it very inconsistent and
> error prone. Moreover the protein of interest is very small (<20 kDa).
> I would appreciate any idea or suggestion in this regards however absurd
> it may sound. As you can imagine, I am at my wit's end myself.
> I was thinking of ELISA lately, but would use of polyclonal antibody be
> a good idea?
> thank and I would appreciate any suggestion on this issue
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
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