Bradford Assay

Introduction

The Bradford assay should be used to quantify total protein within a sample relative to a standard curve. This is important when running proteins on SDS-PAGE/Western blot, where each lane must contain an identical amount of total protein. This way, the intensity of the bands can be compared between different lanes, as standardizing the amount of protein loaded per lane will account for any variation in cell count from which the proteins were derived.

Protocol

Some things to know:

• This protocol uses Bio-Rad products
• This protocol assumes the use of bovine serum albumin (BSA) for the standard curve, which has a linear range of 200 μg/mL to 1000 μg/mL within Bio-Rad's protein assay dye. Linear range may be improved by using other products, but are more expensive than Bio-Rad's.
• If your protein is within a supernatant containing an acid indicator, such as phenol red, this may interfere with the assay. If your protein is within a solution containing phenol red, then ensure that all samples, standards, and blanks are diluted with that media (e.g. Serum free DMEM).
• This protocol assumes all standards, samples, and blanks are run in triplicates.
• Each tube will have extra volume to ensure you have enough for the last replicate.
• Reverse pipetting technique is highly recommended to avoid air bubbles and to ensure accuracy.
• The Bradford Dye is a 5X stock solution.

For a 96 well plate

1. Using a 0.2 μm filter and 15 mL syringe, filter approximately 4 mL of Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford Dye) into a 5 mL Eppendorf tube or 15 mL falcon tube. Let it warm to room temperature.
2. Collect lysate or supernatant that which the protein concentration needs to be calculated for. Keep on ice.
3. In separate 1.5 mL tubes, dilute the sample using ddH2O (or whatever buffer/media is appropriate) into 1:2 and 1:10 dilutions into a total of 600 μL. (NOTE: Do not use the 1:2 diluted sample to prepare the 1:10 tube. Use the original, undiluted sample to prepare the 1:10 dilution)
4. Using a 2 mg/mL BSA stock solution (Bio-Rad), prepare a dilution series using ddH2O (or whatever buffer/media is appropriate) in 1.5 mL tubes: 1 mg/mL, 0.8 mg/mL, 0.6 mg/mL, 0.4 mg/mL, and 0.2 mg/mL. See Figure 1.
5. See Figure 2 for a general plate setup. Using the reverse pipetting technique, pipet 160 μL of blank, standard, and samples into each appropriate well. Avoid creating air bubbles.
6. Spin down the plate at 300 rpm for 1 min to remove bubbles and any liquid on the side of the well.
7. Dump 4 mL of filtered Bradford Dye into a multichannel pipette reservoir. Using a multichannel pipette, pipet 40 μL of dye into each well. If a multichannel pipette is not available, quickly pipet 40 μL of dye into each well using a P200 pipette.
8. Incubate for at least 5 minutes at room temperature.
9. Take off the lid of the plate, and place it into a plate reader/spectrometer.
10. Measure absorbance at 595 nm. Export results to Excel.

Figure 1. General plate layout

Analysis using the standard curve