Enzyme-linked immunosorbent assay
by Karmella Haynes, 2013
Principle: Proteins are 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.
This key feature of this approach is the attachment of proteins directly to the bottom surfaces of the micro wells. A specific protein is detected with a primary and secondary-HRP. The advantage over "sandwich" ELISA is that fewer antibodies are needed, but this method is known to be less sensitive/ accurate. Because of this I recommend this method for artificially over-expressed or abundant naturally expressed proteins.
- Femto ELISA HRP kit (G Biosciences 786-110)
- 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.