Coumassie G is the reagent for Bradford's assay, and Lowry's assay uses the3
Folin's. So, there is not much of a deal there. Your spec reading for the 0
BSA, no matter what it is, should be used as the baseline. In other words, that
IS THE starting point, the zero. The spectrophotometer has to be "Zero'd" with
the 0 BSA tube (which also means the "Water Blank"). I cannot fathom where the
confusion is here! The Lowry assay is prone to interference by a lot of
different things that may be there in your extraction buffer, so, it would be a
a great idea to use the extraction buffer instead of water for the blank
("Buffer Blank").
As someone else also suggested, you may be better of using the modified Lowry
assay called the BCA assay (sold by Pierce). You may also try Bradford's assay
which seems to be best suited for samples containing interfering salts.
Hiranya
Quoting James Wee <jywee at charter.net>:
> I use OD 750 to read cellular protein concentration of a bacteria. I don't
> use Coomassie reagent, instead, I use Folin reagent as the stain. I use 30,
> 60, 90 and 120 µg of BSA to construct the standard curve. It doesn't sound
> right to substract 0.248 from all sample readings. Some of the sample
> readings will go below 0, though the curve goes through 0. Could u explain
> more about over the linear range of detection?
>> Thanks,
> James
>>>
--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
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