Protein assay problems

Alberto mrealpe at ibt.unam.mx
Thu Jan 25 17:47:45 EST 2001


Hi all.  I am following this matter on protein quantification due my own
troubles an I would like to ask about it:

1.  Is there any possibility of ¨managing¨ the interference of triton
X-100 in the lowry method ?. I use to run gradients with triton X-100 and
recently I found a sort of inconsistency in the protein determination
along the fractions.

2.  Among A280, Bradford, Lowry and BCA method, which one would be better
for membrane proteins.  I have noted a lot of modifications of the lowry
method but as I mention before these could not work with my experiments.
Any sugestions ?.

3.  Sucrose gradients fractions have a high level of sucrose in some of
them, is there any problem with this non-reductor sugar for the lowry
method ?.  My dilution is 1:1000.

Regards and thanx in advance for commentaries.  Any recent review on this
matter would be quite appreciated.

Mauricio

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	MAURICIO REALPE
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On Thu, 25 Jan 2001, The Grouchybeast wrote:

> Henk Veldman wrote:
> 
> > The Bio-Rad DC Protein Assay (a variant of Lowry), when done in microtiter
> > plates,  uses 5 ul of sample and will tolerate at least up to 1 % of SDS. It
> > is, however, incompatable with mercaptoethanol and you need a protein
> > concentration in the 0.1 to 1.5 mg/ml range.
> 
> I tried that assay, but a 1:10 dilution of sample was right down at the
> end of the calibration curve.  Actually, I wasn't convinced it wasn't
> just interference in the assay by SDS or Tris.  
> 
> If I used neat protein in the assay, I wouldn't have enough to do any
> replicates of the sample - I like to do triplicates at a minimum.  Plus,
> there would then be >1% SDS in the sample.
> 
> Thanks for the suggestion, though.
> 
> > Henk
> 
> love
> Anna
> -- 
> I see you standing there, far out along the way,
>    I want to touch you but, the night becomes the day.
> I count the words that I am never going to say,
>    And I see you, in midnight blue.    (ELO - Midnight Blue)
> 
> 


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