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

MATERIALS

• Transparent flat-bottom 96-well plate (e.g. ###)
• Bradford reagent (e.g., Sigma B6916-500ML)
• Bovine Serum Albumin (BSA) (e.g., New England Biolabs 10 mg/mL B9001S)

EQUIPMENT

• Biotek Synergy H1 Plate Reader (or similar)

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. Dilute the stock BSA in a new tube to make 50 μL of 1 μg/μL BSA. Example: if the stock BSA is 10 mg/mL, add 5 μL of BSA to 45 μL dH₂O in a fresh tube.
4. 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, see Table 1).
5. Add 5.0 μL of unknown to each remaining tube. Keep track of your samples with good labeling.
6. Transfer 200 μL of the blank (tube one) into the first well in a clear 96-well flat-bottom plate.
7. 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.
8. Use a plate reader to record absorbance at 590 nm (OD 590).

Table 1. Standard sample set-up

Reagent	Tube 1 (0 μg BSA)	Tube 2 (1 μg BSA)	Tube 3 (2 μg BSA)	Tube 4 (4 μg BSA)	Tube 5 8 μg BSA)	Tube 5 (16 μg BSA)
Bradford Reagent	500 μL	500 μL	500 μL	500 μL	500 μL	500 μL
BSA diluted to 1 μg/μL	0 μL	1.0 μL	2.0 μL	4.0 μL	8.0 μL	16.0 μL

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.