Protein assay problems

Emir ekhatipo at NOSPAMmidway.uchicago.edu
Tue Jan 23 18:47:36 EST 2001


>From what you wrote I may suspect that the kit uses nitrocellulose (or other
capable of binding protein) membrane and amido black or a similar dye
(although I did not check their website). It is a pretty old,
pre-spectrophotometric method. Dot blot your protein, same with the standard
at different concentrations, wash blots and apply amido black solution
(something like 0.1 g/100ml in 5% acetic acid, if I am not mistaken). The
visual comparison of the density of color allows you to estimate
concentration of the unknown protein. I guess you could develop such an
assay yourself without buying anything.

Another possibility to consider, try precipitate your protein with 5-10%
(final) trichloracetic acid, spin down the precipitate and redisolve it in
0.1 N NaOH. Use the resuspended protein in any conventional protein assay
(Bradford), but do not forget to use the standard protein dissolved in NaOH,
too. However, I would check if SDS is not getting precipitated by TCA first.

Emir

"The Grouchybeast" <thegrouchybeast at hotmail.com> wrote in message
news:3A6DBD35.B03B9A51 at hotmail.com...
> 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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