Protein assay problems

The Grouchybeast thegrouchybeast at hotmail.com
Tue Jan 23 12:19:49 EST 2001


I am attempting to do Western blots with material from Laser Capture
Microdissection caps.

My problems are as follows:

1. I need to quantitate the protein in order to load equal amounts on
the gel.
2. With the assay kits I have, each cap gives me enough protein to assay
*or* run, but not both.
3. The samples stick quite firmly to the cap so to solubilise the
protein I'm having to use buffer with significant amounts of SDS in it,
which messes up many protein assays anyway.

I am reluctant to try to precipitate out proteins before assaying
because I have so little protein and I'm worried about creating false
variability betwen samples.

I came across the 'Protein dotMETRIC' kit from a company called Geno
Technology.  (http://www.genotech.com).  This kit would seem to solve my
problems because it requires very small amounts of sample and claims to
be tolerant of SDS, b-mercapto and in fact to be useable with samples in
SDS-PAGE loading buffer.  IT claims an accuracy of +/-5%.

Now, if this were true it would be great.  However, the method involves
dotting 2-4ul of 1:10 diluted samples onto a test strip and measuring
the width of the dots to get the protein concentration.  I have never
seen such a method used and have grown wary of uncorroborated claims by
manufacturers.

Does anyone have any experience with this kit or with similar methods? 
I'd also be interested in any other suggestions people might have about
assaying my proteins - I have a max of ~5ul available for assaying from
each sample.

love
Anna
-- 
'It might look like I'm doing nothing, but at the cellular level I'm
really quite busy.'






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