Biology 211 Intro Molecular Cell Biology Laboratory
Bradford Protein Assay Week 9
Purpose: To learn the Bradford procedure for determining protein concentration
on cell extract samples.
References:
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Analytical Biochemistry 72, 248-254
Robyt, J. F. and White, B. J. 1987.
Practice. Brooks/Cole, Monterey, CA.
Biochemical Techniques: Theory and
Background:
Many procedures used in cell biology require knowledge of the protein
content of cells or organelles. We will use the Bradford assay to determine the
amount of protein in a sample of the cell extract we prepared from the NIH 3T3
cells.
The Bradford assay uses a dye, Coomassie brilliant blue G-250, which
has a negative charge and binds with positive charges on the protein. The dye
exists in a red form and a blue form (Amax =595 nm). The red form is the
predominant form in solution and when its negative charge binds to the positive
charges on the protein, it is converted into the blue form. The reaction is highly
reproducible and rapid and is essentially complete after 2 min. with color stability
over 1 hour. At 595 nm absorption is linear over a specific and narrow range of
10-100 ug protein.
Because many proteins have nearly identical response curves, the
method can be applied using a single set of standards. Standard curves are
commonly prepared using bovine serum albumin (BSA). A standard curve
provides a reference for measuring the amount of protein in a solution of
unknown concentration. It is constructed by measuring the absorption of several
known concentrations of protein in the range of 10-100 ug. Then, when a solution
of unknown concentration is measured, its absorbance at 595 nm can be
compared to the standard curve.
The Bradford Assay Lab involves two parts. First, a standard curve will be
constructed using a solution of known concentration of bovine serum albumin
(BSA). Then a sample of the cell extract will be tested for its absorbance in the
assay and its concentration will be determined from the standard curve.
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Procedure: Bradford Assay of Protein
1. The standard curve
a. Obtain a sample of bovine serum albumin (BSA) prepared in distilled water at
a concentration of 100 ug/ml.
b. Label a set of test tubes (13 x 100 mm) from 1-7 using a black Sharpie
marker. Following the chart below fill the tubes sequentially with BSA, water and
Bradford reagent. NOTE: It is important to mix the tubes rapidly and thoroughly
immediately after the dye is added, one tube at a time.
Tube
ddH20 ml
1
BSA @100
ug/ml
ml
0 ml
2.4 ml
Bradford
Reagent
ml
0.6 ml
2
0.1 ml
2.3 ml
0.6 ml
3
0.2 ml
2.2 ml
0.6 ml
4
0.4 ml
2.0 ml
0.6 ml
5
0.6 ml
1.8 ml
0.6 ml
6
0.8 ml
1.6 ml
0.6 ml
7
1.0 ml
1.4 ml
0.6 ml
ug protein*
X-axis
A595
Y-axis
0 (blank)
0 ug
* calculate ug protein by multiplying BSA concentration (100 ug/ml) by the
volume used (ml)
Use the tube #1, the BLANK, to set the absorbance at 595 nm to zero. Measure
the A595 of each of the other tubes. Write the values in the chart above. Plot
your data on graph paper, or using the excel spreadsheet provided, with ug
protein on the X axis and A595 on the Y axis. Show your graph to the instructor.
2. Assay of the cell extract
In this portion of the laboratory you will determine the concentration of the cell
extract you prepared last week. To break the cells open, the cells were frozen at
-80C, then thawed. This cycle was repeated several times to create a "freezethaw" extract.
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a. Obtain your tubes of extract and keep on ice.
b. Label a test tube for each tube of extract you wish to test. Add the
components in the order protein sample, water, Bradford reagent and mix
thoroughly.
Tube
Cell extract
Sample
0.1 ml
ddH20
Bradford
reagent
(ml)
2.3 ml
ug protein
A595
(from std.
Curve)
0.6 ml
c. Determine the absorbance reading for the cell extract sample. Use the
standard curve to determine the the amount of protein (in ug) for the sample. If
the absorbance reading falls outside the linear range of the standard curve,
repeat the Bradford Assay using less sample 10-50 ul (=0.01 to 0.05 ul).
d. The remainder of the cell extract should be kept on ice to be returned to the 80 C freezer for next week's lab.
e. For next week's lab, you will need to determine a volume of cell extract that
contains 10-20 ug protein. We will have access to micropipets that can measure
smaller volumes (in microliters).
1000 ul = 1 ml
100 ul = 0.1 ml
10 ul = 0.01 ml
f. Hand in your completed tables, your standard curve and your
calculations (of the protein concentration in the cell extract and the volume
to use for electrophoresis), by next week’s lab. This assignment is worth
20 points.
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