Bradford Assay Experimental Protocol

Bradford Assay Materials & Reagents

This assay is a relatively simple process. If you need direct access to our data processing application rather than a review of the protocol, then login to the Bradford Assay eForm. Otherwise, please continue reviewing the Bradford assay experimental procedure. The following equipment, materials, and reagents are required:

• Refrigerator (4°C)
• Freezer (non-frostfree,-20°C)
• Ice Bucket w/ice
• Vortex mixer
• Timer
• Gloves
• Sharpie/Permanent ink marker
• Filter paper (e.g. Whatman #1)
• Funnel
• Graduated Cylinder
• Graduated Pipettes/Pipetter
• Manual Pipetter w/Tips
• Ehrlenmeyer Flask

• Beaker
• Test tube/Cuvettes (or Multiwell Plates)
• Test tube/Cuvette Rack
• Stirrer
• Stirring Bar
• Spectrophotometer
• Coomassie^® Brilliant Blue dye
• Protein Standard (i.e. BSA or IgG)
• Protein Samples of Unknowns
• ddH₂O
• Bradford Assay eForm Application

Bradford Assay Procedure

1. Remove refrigerated Coomassie Brilliant Blue (Bradford dye concentrate) and frozen standards and samples.
2. Place frozen standards and samples in ice bucket.
3. Remove aliquots from standards and samples and let thaw as needed.
4. Turn on spectrophotometer to allow the device to warm-up and equilibrate.
5. Agitate Bradford concentrate to mix and, using graduated cylinder and/or pipetter, withdraw an amount in accordance with manufacturer's directions.
6. Place a beaker onto a stirrer and add a stirring bar.
7. Dump Bradford concentrate into beaker and add ddH₂O in a ratio in accordance with manufacturer's directions while stirring (1:4 is typical [1.0 ml dye + 4.0 ml ddH₂O, for example]).
8. Secure a paper filter into the neck of an Ehrlenmeyer flask and gravity filter the diluted Bradford reagent.
9. Add a stirring bar to the Ehrlenmeyer flask and continue mixing on the stirrer.
10. After determining the total number of test tubes or cuvettes required to produce duplicates (or triplicates), label each tube according to your prepared grid and place into rack.
11. Pipet standard curve protein samples and unknown samples into the appropriate test tube.
12. Aliquot increasing titers of Bradford dilutions and pipet them into racked cuvettes.
13. Vortex samples briefly to ensure good mix in suspension.
14. Incubate at room temperature for at least 5 minutes (1 hr max). Absorbance will increase with time.
15. Set a baseline for the spectrophotometer by placing a "blank" sample (i.e. BSA or IgG) into the device and calibrating by zeroing.
16. Measure absorbance at 595 nm (or other desired wavelength) for each standard curve sample and experimental unknown sample in sequence and without extended interruptions.
17. Record relevant experimental summary and data; enter into Bradford Assay eForm Application.
18. Create, save, print, or send report.