Bradford Protein Assay For Estimation of Total Protein Content
The Bradford Protein Assay is a widely used assay for the estimation of total protein concentration. The Bradford Protein Assays uses a protein of the researcher's choice to develop a standard curve to which all subsequent measurements are based. The procedure involves the reaction of a color reagent with the specific amino acids of the protein. If a particular protein has abnormally less or more of the particular amino acids, it could give a lower or higher value for the amount of protein. Using this method you will be able to determine the amount of protein in your research sample. If you are working with an enzyme, by knowing the total amount of protein in the sample, you can determine the percentage of ENZYME per amount of protein.
For our laboratory exercise, Bovine Serum Albumin (BSA) will be used in the preparation of a standard curve. The amount of protein in your research sample will then be compared to the BSA standard curve. READ THROUGH THE FOLLOWING GENERAL PROCEDURES BEFORE BEGINNING YOUR WORK.
PREPARATION OF THE STANDARD CURVE
(1) Set up the Spectrophotometer before you start the Standard Curve.
(2) Turn on the visible (325 nM to 900 nM) light source by pressing the "VIS" key and the ultraviolet light source (190 nM - 325 nM) by pressing the "UV" key. Select the Absorbance mode. While the spectrophotometer is warming-up, prepare the BSA and Bradford Reagent for calculation of the standard curve.
Preparation of the BSA for use as the
standard protein: use of the
extinction coefficient
(3) Obtain 100 ml of deionized water in
a 200 or 250 ml beaker. Add a magnetic stir bar, and place on a stirring
plate, mix at a somewhat slow speed.
(4) Weigh out 100 mg of BSA, and slowly stir the BSA into the 100 ml of deionized water.
(5) Stir several minutes (may require slight heating to completely dissolve the BSA, however, do not boil or over heat).
(6) Transfer about 2 ml of this BSA sample to a quartz spectrophotometer cuvett. Take an Absorbance reading at 280 nm. Record this value. Note: quartz cuvetts are very expensive, treat them gently.
(7) Divide the Absorbance value obtained for the 1 mg/ml BSA by 0.667 (extinction coefficient of BSA) to obtain the actual concentration. The extinction coefficient of BSA is the actual reading that should be obtained if exactly 1.0 mg/ml solution was prepared. However, most solutions, unless made with the utmost accuracy, contain error.
Absorbance (280 nm) = _______ - 0.677 = __________ mg/ml
This gives the true concentration of BSA in solution. Use this concentration to determine the actual amount of BSA in the chart below.
(8) Use this calculated BSA concentration value to determine the exact amount of protein in sample volumes of 10 ul, 20 ul, 30 ul,... 100 ul. These values will be used to set up the STANDARD CURVE.
ul of BSA
mg of BSA
ug of BSA
10 ul
_________ _________
20 ul
_________ _________
30 ul
_________ _________
40 ul
_________ _________
50 ul
_________ _________
60 ul
_________ _________
70 ul
_________ _________
80 ul
_________ _________
90 ul
_________ _________
100 ul
_________ _________
Preparation of the standard curve:
(9) Set up 10 test tubes (15-20 ml size).
(10) Label the test tubes "10 ul, 20 ul, 30 ul,...100 ul".
(11) Fill test tube with 5 ml of Bradford Reagent. NOTE: the Bradford Reagent that we get is only a stock solution. Read the label on the bottle to determine how to dilute this stock solution for the following work.
(12) Using the standard BSA solution you made above, add the BSA to the appropriate test tubes in 10 ul increments (10 ul, 20 ul, 30 ul, ... 100 ul). Note that by using the extinction coefficient, you actually know the amount of BSA in each 10 ul increment.
(13) Cover the top of the test tube with Parafilm and mix by slowly inverting. When mixing the samples it is important not to create bubbles. Proteins, in their pure forms, tend to foam. This foaming action will damage the proteins, possibly giving you an error in the absorbance readings.
(14) Transfer about 2 ml of this Bradford-BSA solution to separate plastic cuvetts.
(15) Take readings at 595 nm.
ul of BSA
ug of BSA
Absorbance at 595 nm
10 ul
_________
_________
20 ul
_________
_________
30 ul
_________
_________
40 ul
_________
_________
50 ul
_________
_________
60 ul
_________
_________
70 ul
_________
_________
80 ul
_________
_________
90 ul
_________
_________
100 ul
_________
_________
(16) Plot the above data with
the ug of BSA on the x-axis and absorbance on the y-axis. This should
give you a straight line. Use this standard curve to help calculate
the amount of protein in your sample as directed below.
Determination of protein concentration
in an unknown sample:
(17) Now you will set up three additional
test tubes that will be used in the determination of protein concentration
in your samples. Label these test tubes 5 ul, 10 ul, and 20 ul, then
fill each of the three test tubes with 5 ml of Bradford Reagent.
(18) Add 5, 10, and 20 ul of your undiluted protein sample to tubes, respectively. Cover the tubes with Parafilm and mix by inversion.
(19) Transfer about 2 ml of this Bradford-BSA solution to separate plastic cuvetts.
(20) Take readings at 595 nm. Using your standard curve, find the absorbance value on the y-axis. Read over to the standard curve line, then down to the x-axis. The point of intersection on the x-axis is the amount of protein in your sample.
NOTE: At this point you may need to do this portion of the procedure several times if your absorbance readings do not fall within the values on the standard curve. If the absorbance readings are too high, you will need to dilute a portion of your sample and do the readings again. Remember to calculate in the dilution factor. If the absorbance readings are too low, you will need to increase the amount of sample.
Calculate the amount of protein per volume
of your protein sample (ug/ul). This is easily done by dividing the
ug of protein by the ul of sample. Average the three ug/ul values
to obtain the amount of protein in the Extract per 1 ul.