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.
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
> 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)