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Protein assay problems

engelbert_buxbaum4 at my-deja.com engelbert_buxbaum4 at my-deja.com
Wed Jan 24 03:25:50 EST 2001


In article <3A6DBD35.B03B9A51 at hotmail.com>,
  The Grouchybeast <thegrouchybeast at hotmail.com> wrote:
> I am attempting to do Western blots with material from Laser Capture
> Microdissection caps.

> 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.


This depends on the method you use. Chloroform/Methanol
precipitation (Wessel + Fl"ugge) is quite reliable, and if you follow
that with the BCA protein determination (check the Pierce catalogue) the
whole thing is quite sensitive (especially in microcuvettes, were you
can work with 50-100 ul total volume at 10 mm path length)


>
> 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.

Check the publication by Mahoney et al (Anal. Biochem. 248 (1997) 182-4)
for a way to do this more quantitatively.


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