Western problem

Jayakumar r.jayakumar at roswellpark.org
Thu Jul 3 08:45:42 EST 2003


Hi all..
    I am having a very strange problem with my western blots.  Here is what
I do.  I have these mitochondrial suspensions of known protein conc. (in a
buffer containing 70mM sucrose, 220mM mannitol, 2mM Hepes pH 7.44, 0.5mg/ml
BSA and other protease inhibitors).  I suspend my mito pellet by vortexing
(I am not bothered about its integrity) and then by triturating it with a
P-200 pipettman, so as to homogeneously suspend it.  I then go ahead and
prepare the sample with the required amount of protein with 4X Laemmli
buffer.  I have to load them in dulicates and in several concentrations.  I
make a master mix of the protein with the Laemmli buffer, boil it, spin it
briefly and vortex it (since I want to load everything including the
precipitates whcih may have membrane proteins).  Then I load the required
volume from this master mix so as to get the required concentrations in
duplicates.
     Strangely I dont get uniformity in band intensity between the same
concentrations.  Some bands dont appear at all, while some are very intense
even though all of them are loaded with the same volume that should have the
same protein concentrations.
     I tried extracting the mito with a buffer containing NP-40 (IGEPAL) and
then doing the same thing.  I am still getting non-uniform band intensity
(Some are light and some are dark), for the same protein loads.
     Please let me know what I am doing wrong here.  I am at my wits end.
     Any help would be gratefully acknowledged.
regards
Jai


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