- After performing the Bradford assay, make a graph by plotting the spectrophotometer absorbance readings vs. the concentration of each of the BSA standards. Draw a straight line through the points. If all the points do not lie on a line, draw a line of best fit. A sample standard curve is shown in figure 3.1 above.
- Using the spectrophotometer readings(s) for your unknown(s), determine the concentration (in µg/ml) for your unknown sample(s) from the standard curve. Suppose that your sample had an absorbance of 0.61. If you look at the graph above, this absorbance value corresponds to a protein concentration of 15 µg/ml.
- The concentration that you read from the graph in step 2 is the protein concentration of your diluted sample.
- Now you need to account for any dilutions that you made. If you took 20 µl of undiluted sample and 780 µl of water to make the 800 µl, that dilution is 20/800 or 1/40. You will therefore multiply the answer by 40. (If you made a 1/100 dilution, you will need to multiply by 100, etc., etc.)
- If your dilution factor was 40, your original undiluted sample contains 600 µg/ml or 0.6mg/ml of total protein. (If your dilution factor was 100, then your undiluted sample contains 1500 µg/ml or 1.5 mg/ml.)
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