Haynes:Bradford: Difference between revisions
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# Label enough 1.5 mL eppendorf tubes for one blank (1) , five standard samples (2-6), and all of your unknown samples (7-...''n''). | # Label enough 1.5 mL eppendorf tubes for one blank (1) , five standard samples (2-6), and all of your unknown samples (7-...''n''). | ||
# 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. | # 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. | ||
# 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). | # 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 dH<sub>2</sub>O in a fresh tube. | ||
# 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). | |||
# Add 5.0 μL of unknown to each remaining tube. Keep track of your samples with good labeling. | # Add 5.0 μL of unknown to each remaining tube. Keep track of your samples with good labeling. | ||
# Transfer 200 μL of the blank (tube one) into the first well in a clear 96-well flat-bottom plate. | # Transfer 200 μL of the blank (tube one) into the first well in a clear 96-well flat-bottom plate. | ||
# 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. | # 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. | ||
# Use a plate reader to record absorbance at 590 nm (OD 590). | # Use a plate reader to record absorbance at 590 nm (OD 590). | ||
Table 1. Standard sample set-up | |||
{| {{table}} | |||
|- | |||
| BSA || Vol. Bradford Reagent || Tube 1 (0 μg BSA) || Tube 2 (1 μg BSA) || Tube 3 (4 μg BSA) || Tube 4 (8 μg BSA) || Tube 5 (16 μg BSA) || | |||
|- | |||
| NEB B9001S 10 mg/mL || 500 μL || 0 μL || 1.0 μL || 2.0 μL || 4.0 μL || 8.0 μL || 16.0 μL | |||
|} | |||
Revision as of 12:40, 15 September 2014
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
- Label enough 1.5 mL eppendorf tubes for one blank (1) , five standard samples (2-6), and all of your unknown samples (7-...n).
- 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.
- 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.
- 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).
- Add 5.0 μL of unknown to each remaining tube. Keep track of your samples with good labeling.
- Transfer 200 μL of the blank (tube one) into the first well in a clear 96-well flat-bottom plate.
- 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.
- Use a plate reader to record absorbance at 590 nm (OD 590).
Table 1. Standard sample set-up
| BSA | Vol. Bradford Reagent | Tube 1 (0 μg BSA) | Tube 2 (1 μg BSA) | Tube 3 (4 μg BSA) | Tube 4 (8 μg BSA) | Tube 5 (16 μg BSA) | |
| NEB B9001S 10 mg/mL | 500 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)
- Subtract the blank OD 590 value from all other values.
- Plot a standard curve (using Excel) with BSA concentration (x-axis) vs. Absorbance at 590 nm (y-axis). See this example.
- Add a line of best fit (not a curve) and display the equation.
- 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.
