Bio-Rad DC™ Protein Assay - Manual (Page 7)

1. The buffer that I normally use
is not listed in the reagent
compatibility list. How will I
know if it interferes with the
assay?
2. My sample is a mixture of
proteins. Which standard
should I use for the standard
curve?
3. Is any sample preparation
required?
4. May I use a wavelength other
than 750 nm?
5. May I store the reagents in the
refrigerator?
It is best to run two standard curves: One
with protein in the same buffer as your
sample and one with protein in water and
then plot a graph of protein concentra-
tion vs. absorbance. If the buffer does
not interfere, the two graphs of the stan-
dard curve will have identical slope.
Partial interference can be compensated
for by adding the buffer or interfering
component to the standard curve for the
actual protein assay.
In any protein assay, the best protein to
use as a standard is a purified preparation
of the protein being assayed. In the
absence of such an absolute reference
protein, one must select another protein
as a relative standard. The best relative
standard to use is one which gives a color
yield similar to that of the protein being
assayed.
Any purified protein can be selected as a
reference standard if only relative pro-
tein values are desired. Bio-Rad offers
two standards: bovine gamma globulin
(Standard I, catalog number 500-0005)
and bovine serum albumin (Standard II,
catalog number 500-0007).
In general, no. However, the protein must
be solubilized. (The sample can not be a
suspension or an unfiltered homogenate.)
Yes. Absorbance can be measured at 650-
750 nm.
Yes, but all components must be warmed
to 25-30 °C prior to use. Reagent S will
develop a precipitate during cold room
storage. Warming to 37 °C for 5 minutes
will dissolve the precipitate.
5
Section 8
Troubleshooting Guide
LIT448D 7/21/98 1:16 PM Page 5