Thermo Scientific Pierce Protein Assay Technical Handbook Version 2

Thermo Scientific Total Protein Assays
The Modified Lowry Protein Assay offers all of the advantages of
the original reagent introduced by Oliver Lowry in 1951 in a single,
stable and ready-to-use reagent.
If the samples to be analyzed contain one or more detergents
(at concentrations up to 5%), the BCA Protein Assay is the best
choice. If the protein concentration in the detergent-containing
samples is expected to be very low (< 20µg/mL), the Micro BCA
Protein Assay may be the best choice. If the total protein concentration
in the samples is high (> 2,000µg/mL), sample dilution
can often be used to overcome any problems with known
interfering substances.
Sometimes the sample contains substances that make it incompatible
with any of the protein assay methods. The preferred method
of dealing with interfering substances is to simply remove them.
We offer several methods for performing this function, including
dialysis, desalting, chemical blocking and protein precipitation
followed by resolubilization. This handbook focuses on the last
two methods. Chemical blocking involves treating the sample with
something that prevents the interfering substance from causing
a problem. Protein precipitation causes the protein to fall out of
solution, at which time the interfering buffer can be removed and
the protein resolubilized. The chemical treatment method, like that
used in the BCA Protein Assay – Reducing Agent Compatible, or
the Pierce 660nm Protein Assay is generally preferred because,
unlike protein precipitation, resolubilization of potentially hydrophobic
proteins is not involved.
Selection of a Protein Standard
Selection of a protein standard is potentially the greatest source of
error in any protein assay. Of course, the best choice for a standard
is a highly purified version of the predominant protein found in the
samples. This is not always possible or necessary. In some cases, all
that is needed is a rough estimate of the total protein concentration
in the sample. For example, in the early stages of purifying a protein,
identifying which fractions contain the most protein may be all that
is required. If a highly purified version of the protein of interest is not
available or if it is too expensive to use as the standard, the alternative
is to choose a protein that will produce a very similar color
response curve with the selected protein assay method. For general
protein assay work, bovine serum albumin (BSA) works well for a
protein standard because it is widely available in high purity and is
relatively inexpensive. Although it is a mixture containing several
immunoglobulins, bovine gamma globulin (BGG) also is a good choice
for a standard when determining the concentration of antibodies,
because BGG produces a color response curve that is very similar to
that of immunoglobulin G (IgG).
For greatest accuracy in estimating total protein concentration in
unknown samples, it is essential to include a standard curve each
time the assay is performed. This is particularly true for the protein
assay methods that produce nonlinear standard curves. Determination
of the number of standards and replicates used to define
the standard curve depends upon the degree of nonlinearity in the
standard curve and the degree of accuracy required. In general,
fewer points are needed to construct a standard curve if the color
response is linear. Typically, standard curves are constructed using
at least two replicates for each point on the curve.
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.
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