Bradford Protein Assay   

Abstract

Materials and Reagents

1. Bovine Serum Abumin (BSA) (Sigma-Aldrich)
   
2. Coomassie Brilliant Blue G-250 (Sigma-Aldrich, catalog number: 27815 )
   
3. Methanol
   
4. Phosphoric acid (H₃PO₄)
   
5. Bradford reagent (see Recipes)
   

Equipment

1. Spectrophotometer (Tecan)
   
2. Whatman #1 paper (Whatman)
   

Procedure

1. Standard assay procedure (for sample with 5-100 µg ml^-1 protein)
   1. Prepare five to eight dilutions of a protein (usually BSA) standard with a range of 5 to 100 µg protein.
      
   2. Dilute unknown protein samples to obtain 5-100 µg protein/30 µl.
      
   3. Add 30 µl each of standard solution or unknown protein sample to an appropriately labeled test tube.
      
   4. Set two blank tubes. For the standard curve, add 30 µl H₂O instead of the standard solution. For the unknown protein samples, add 30 µl protein preparation buffer instead. Protein solutions are normally assayed in duplicate or triplicate.
      
   5. Add 1.5 ml of Bradford reagent to each tube and mix well.
      
   6. Incubate at room temperature (RT) for at least 5 min. Absorbance will increase over time; samples should incubate at RT for no more than 1 h.
      
   7. Measure absorbance at 595 nm.
      
   
   
2. Microassay procedure (<50 µg ml^-1 protein):
   1. Prepare five standard solutions (1 ml each) containing 0, 10, 20, 30, 40 and 50 µg ml^-1 BSA
      
   2. Pipet 800 μl of each standard and sample solution (containing for <50 µg ml^-1 protein) into a clean, dry test tube. Protein solutions are normally assayed in duplicate or triplicate.
      
   3. Add 200 μl of dye reagent concentrate to each tube and vortex.
      
   4. Follow the procedure described above for the standard assay procedure.
      

Recipes

1. Bradford reagent
   Dissolve 50 mg of Coomassie Brilliant Blue G-250 in 50 ml of methanol and add 100 ml 85% (w/v) phosphoric acid (H₃PO₄).
   Add the acid solution mixture slowly into 850 ml of H₂O and let the dye dissolve completely (note: Do not add H₂O into the acid solution).
   Filter using Whatman #1 paper to remove the precipitates just before use.
   Store in a dark bottle at 4 °C.
   

Acknowledgments

This work was done in the Andrew Binns Lab in the Department of Biology at University of Pennsylvania, USA and supported by National Science Foundation grants MCB 0421885 and IOS-0818613.

References

1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
   
2. Stoscheck, C. M. (1990). Quantitation of protein. Methods Enzymol 182: 50-68.