by Karmella Haynes, 2012
Principle: Protein is captured on the bottom of a micro well plate, either by direct binding or by a conjugated antibody "trap". A second antibody is added to detect one specific type of protein. A counter-stain antibody (usually HRP-conjugated) is used to generate visible signal, which is proportional to the number of proteins. Normalization (e.g., using the number of cells per lysate sample, and a purified protein with known concentration if you're fortunate to have one available) can be used to calculate proteins per cell.
- 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 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).
- 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).
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.