PROVOST'&'WALLERT'RESEARCH!
Investigating the Biochemistry &
Cellular Physiology of NHE1
EST. 1998
Bradford (BioRad)
Protein Assay
1.5 ml Protocol
!
June 2015
1
Theory and Introduction: The determination of protein concentration is frequently required in biochemical work.
Several methods are available, each having features that suit it to a particular use. There are many reasons to
conduct a protein assay. During a purification of a protein, you need to know how pure your sample is by
determining the amount of enzymatic activity vs. protein concentration. When comparing samples on an SDS-
PAGE gel or using antibody for a Western blot, you need to add the same amount of protein from various
preparations to obtain true results. Most protein assays take advantage of a reaction between a reagent dye and
the protein of interest that will shift or increase the absorbance of a particular wavelength. Generally, the more
protein in a sample, the higher the absorbance. BUT remember the assay must remain linear and follow Beer’s
Law! If the absorbance is higher than 1.5 absorbance units (AU), then look very close at your data before using
it!
Bradford reagent
(we use the reagent prepared by BioRad Protein Assay Solution) uses Coomassie blue G-250.
Without protein, the solution is red-brown in its acidic solution. When protein binds, the pKa of the dye shifts
causing the dye to become blue. The dye is measured at 595 nm. Bradford dye is easy to use, as well as fast
and sensitive, but several compounds can interfere with the assay (
see the pdf file for the BioRad Protein Assay
on the class webpage
).
Standard curve
:
Learning how to prepare and use a standard curve is an incredibly important skill that all
employers and professional schools expect you to know
. Thus you will be conducting several standard curves
while doing a protein assay throughout this semester. A protein assay consists of two main components: the
standard curve and the unknowns. Bradford and Lowry protein assay reagents results in a change in absorbance
when protein is present. How does this absorbance relate to the actual protein concentration? To know
determine the actual concentration of a protein a standard curve is required. A standard curve is a plot of
absorbance vs. a varying amount of some known concentration of protein. Then you will take absorbance readings
in parallel with unknown protein sample (in this case the fractions or pools of your purification).
A common method to prepare a standard curve is to prepare various known protein concentrations as standards.
As long as the volume of the standard samples and the unknown samples are the same the final concentration of
the unknown is directly calculated from the least squares line of the standard curve. Of course, you have to
correct for any dilution of your sample that may be needed to achieve an absorbance that falls within your
standard curve.
Always run samples in duplicate or triplicate and use the averages in the graph. If a sipper is used for reading
samples, it is a good idea to read those samples with less protein content first to reduce error arising from
reagent dye carryover in the cuvette as a result of incomplete rinsing.
Depending on the method, proteins generally vary in the reaction with the color dye in the different protein
assays. Therefore, it is important to denote what the specific control or reference protein was used to make the
standard curve. Two common proteins used for standard curves are bovine serum albumin (BSA) and an
immunoglobin (IgG). These two proteins have different amino acid compositions, which lead to a different
standard curve and a slight difference in the final determination of the unknown protein concentration. Because
the color development is dependent on the amino acid composition of the protein and the presence of a
prosthetic group (especially carbohydrate) which can also influence the protein assay, a purified sample the
protein being assayed or a closely related protein is a preferred standard.
