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Bradford assay is a protein quantification protocol developed by Marion Mckinley Bradford in 1976. It gives an easy was to estimate protein concentration of a solution using a standard curve generated by the use of known concentrations of a protein. First, you need to prepare a standard calibration curve of a known protein using various predefined concentrations and then you can use the linear regression equation generated from the standard curve to determine the concentration of your test sample.
BSA standard calibration curve
Prepare 50 ml of 1 mg/ml stock solution of a standard protein (the most commonly used protein is bovine serum albumin or BSA) in distilled water. From the working solution, prepare 10 different solutions with the concentration ranging from 10 µg to 100 µg and maintain the total volume 100 µl with distilled water.
You should also prepare one control in which there is no BSA present but it contains only the buffer solution in which protein sample is dissolved. Preparing standard calibration curve is an essential part of the protein quantification by Bradford assay.
Preparation of Bradford reagent
Bradford reagent is a solution of Â Coomassie Brilliant Blue (CBB-G250) prepared in a phosphoric acid, ethanol, and distilled Â water. To prepare the Bradford reagent stock solution, take 100 mg CBB-G250 and dissolve it in 50 ml of 95% ethanol, using a volumetric flask of 50 ml.
After complete dissolution of the CBB-G250 in ethanol, add 100 ml of 85% (w/v) phosphoric acid and then add 50 ml of distilled water. Then filter the solution (which is 200 ml now) with number-1 Whatman filter paper and store it in a brown bottle to be used in future as a stock solution. During Bradford assay, you should dilute the stock solution of the Bradford reagent by five times using distilled water (in 1:4 ratio).
Steps of Bradford Assay
- Turn on the Spectrophotometer and allow it to warm up.
- After warm up, set the wavelength to 595 nm.
- Take 1oo µl of BSA working solutions of 10 different concentrations 10 µg to 100 µg in a set of empty test tubes and label them as BSA-1 to BSA-10.
- Add 100 µl of buffer solution in a test tube and label it as a control.
- Take 100 µl of protein sample of unknown concentration in a test tube and label it as a sample. In the same way, take 100 µl of diluted protein sample (2 x dilution) in a test tube and mark it as a 0.5 X Sample.
- Add 5 ml of Bradford reagent (working reagent that is already diluted by five times) in each of the test tubes and allow them for five minutes of incubation.
- After five minutes of incubation, take the control and set the absorbance into auto zero in the spectrophotometer.
- Take the absorbance of each of the standard BSA solutions starting from BSA-1 (10 µg) to BSA-10 (100 µg) and record their absorbance units.
- Now, take the absorbance of the protein samples starting with diluted one and record the absorbance unit.
- Now, prepare a standard curve by plotting a graph of concentration (µg) in Y-axis versus absorbance in X-axis and determine the linear regression equation of that graph.
- Using that linear regression equation (y=mx+c), calculate the concentration of the protein samples of unknown concentration using their absorbance units.
- The concentration of the protein sample that you get will be in µg/5.1 ml. So convert the unit to µg/ml to get the final concentration of the protein sample.