Bradford assay

Assay Principle

The dye Coomassie brilliant blue G-250 binds to proteins (primarily to arginine, lesser to lysine, histidine, tyrosine, tryptophane and phenylalanine residues) under acidic conditions. This creates a blue color with an absorbance maximum at 595nm. Absorbance is proportional to protein amount. From absorbance values of a range of known concentrations of a reference protein, a standard curve can be created, from which protein concentration can be calculated. 

Materials

• transparent 96 well plates (flat or U-shaped) or cuvettes
• 5x Bradford dye concentrate:
  • dissolve 100 mg Coomassie Brilliant Blue G250 in 50 ml 95% ethanol
  • add 100ml concentrated phosphoric acid
  • add H₂O to a final volume of 200ml
• Albumin with known concentration (e.g. 2ml/ml from glass ampoule)
• plate reader or other photometer

Protocol

small volume 96 well version example: 

1. If possible, use the same buffer or water for standards and dilutions
2. prepare standard curve:
   1. pipet 10µL of albumin standard (2mg/ml) into well A1 and A2
   2. fill wells B1-2 to H1-2 with 5µL buffer or water (preferably the same as the unknowns sample)
   3. take 5µL from A1 and fill into B1, mix well by pipetting up and down (while avoiding bubbles) and transfer 5µL from B1 to C1, mix and transfer to D1, etc. continue until G. 
   4. Repeat step 1c for Column 2 
   5. H1 and H2 contains buffer only (c=0)
3. prepare sample dilutions
   1. pipet 10µL of the unknown sample into wells A3 and A4
   2. fill remaining wells with 5µL/well and perform 2-fold serial dilution as described for the standard curve.

      Example plate layout:

      	1	2	3	4	5	6	7	8	9	10	11	12
      A	2 mg/ml	2 mg/ml	Sample 1 undiluted	repl.
      B	1 mg/ml	1 mg/ml	Sample 1 dilution factor 2
      C	500 µg/ml	500 µg/ml	Sample 1 dilution factor 4
      D	250 µg/ml	250 µg/ml	Sample 1 dilution factor 8			etc.
      E	125 µg/ml	125 µg/ml
      F	62,5 µg/ml	62,5 µg/ml
      G	31,25 µg/ml	31,25 µg/ml
      H	0	0
      	Albumin Standard		unknown samples

4. dilute 5x Bradford dye solution 5 fold in H₂O to gain 1x concentrated working solution. (filter if precipitates occur)
5. add 250µL dye solution per well to all standards and samples using the 8-channel pipette. Be careful not to generate foam and bubbles
6. mix and allow the color to develop for 5 minutes but no longer than 30 min.
7. Read absorbance in the microplate reader at 595nm
8. Plot average absorbance values of the replicates versus concentration. Create a linear standard curve.
9. use software interpolation or the linear equation to calculate the concentration of the unknown samples.

Example:

Remarks

• not all chemicals are compatible with Bradford assay 
• 1 test in duplicate gives you a good estimate, the exact values depend on many factors, but technical and handling errors will get smaller if you average more replicates. 
• values are more reliable when the standard with a known concentration is more similar to the unknown sample (which means you can use other proteins as standard as well, best if you have the same with a known concentration)
• Glycoproteins may be shielded from Coomassie dye and appear less than they actually are, the same is true for polymers
• make sure to avoid bubbles and foaming of protein samples, if you have bubbles, try destroying them with a needle before measurement.
• diluted unknowns help to judge the accuracy and the detection range of the test. If you backcalculate the concentration of the sample x dilution factor and get the same values, measurement is valid and can be used to further average the result.