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Bradford Protein Assay using the Pierce Reagent

1. Label a series of cuvettes 0, 0.2, 0.4, 0.6, 0.8 mg/ml. To each add 1 ml Bradford Assay Reagent (stored in the refrigerator). Add 0, 2, 4, 6, or 8 microlitres of 2 mg/ml Bovine Serum Albumin Fraction V (stored at –20˚C). Mix by covering with parafilm and inverting several times.

2. Prepare two to four cuvettes for each sample. To each, add 1 ml of Bradford Assay Reagent. Add an appropriate amount of sample to the first cuvette based on the expected protein concentration: for example, if the expected concentration is around 1 mg/ml, add about 10 microlitres, or if it is around 5 mg/ml, add around 2 microlitres. Mix the cuvette as described above. If the colour is less intense than the 0.2 mg/ml standard, add some more sample and mix again, keeping track of the total amount of sample added. If the colour is more intense than that of the 0.8 mg/ml standard, discard the cuvette and add a smaller amount of sample to the next one. Try to get 2 to 4 cuvettes in the standard curve range for each sample.

3. Set a spectrophotometer to 595 nm and zero it with the 0 mg/ml standard. Read the absorbance at 595 nm of each standard and sample. Draw a standard curve and use this to estimate the protein concentration in each sample. Calculations are based on a sample volume of 20 microlitres, so multiply by an appropriate dilution factor. For example, if 10 microlitres was used, multiply by 2, or if 4 microlitres was used, multiply by 5. Average the results for each sample to get the protein concentration, and if you have three or more cuvettes, work out the standard error (= population estimate standard deviation divided by [number of measurements minus one]).

Safety Notes: The Bradford assay reagent contains methanol. Wear gloves, and optionally eye protection when dispensing it.

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