Haynes:Bradford

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Bradford Assay
by Karmella Haynes, 2012

Principle: The dye in the Bradford reagent turns from brown to blue in the presence of protein. The color change is proportional to the protein concentration. See http://en.wikipedia.org/wiki/Bradford_protein_assay

Procedure

1. Label enough 1.5 mL eppendorf tubes for one blank (1) , five standard samples (2-6), and all of your unknown samples (7-n).
2. Add 500 μL Bradford Reagent to each tube. You will add protein to these later, and ignore the negligible change caused by additional protein volume.
3. Add a BSA standard protein solution* to tubes 2 (1μg BSA), 3 (2μg BSA), 4 (4μg BSA), 5 (8μg BSA), and 6 (16μg BSA). (*Note, use the appropriate volume based on the concentration of your stock BSA).
4. Add 5.0 μL of unknown to each remaining tube. Keep track of your samples with good labeling.
5. Transfer 200 μL of the blank (tube one) into the first well in a clear 96-well flat-bottom plate.
6. Do the same for the others, using new wells, but be sure to mix by pipetting up and down before transferring 200 μL of sample to the 96-well plate.
7. Use a plate reader to record absorbance at 590 nm (OD 590).

What to do with your data: calculate unknown protein concentration(s)

1. Subtract the blank OD 590 value from all other values.
2. Plot a standard curve (using Excel) with BSA concentration (x-axis) vs. Absorbance at 590 nm (y-axis). See this example.
3. Add a line of best fit (not a curve) and display the equation.
4. Solve the equation for x. Substitute y with the background-subtracted OD 590 for the unknowns.
   Protein concentration of the unknown = x μg/ 5.0 μL.