The Bradford assay (Ref. Bradford, M. M. (1976) Anal. Biochem. 72, p248)
The Coomassie Brilliant Blue G-250 dye binds selectively to arginine and aromatic residues, and this binding is accompanied by a shift in absorbance maximum from 470nm to 595nm. The assay is fast, inexpensive and sensitive, and tolerates a wide range of buffers. The Bradford assay is, however, protein dependant, non-linear and detergent incompatible. Expedeon has developed a detergent compatible assay solution, BradfordUltra, which removes the requirement for detergent-solubilised protein to be precipitated before use.
Note! The Coomassie dye binds to quartz, so it is advisable to use glass or plastic cuvettes.
PROTOCOL FOR EXPEDEON’S BRADFORDULTRA
• Mix the BradfordUltra Reagent solution immediately before use by gently inverting the bottle several times (Do not shake the bottle to mix the solution). Remove the amount of reagent needed and equilibrate it to room temperature before use.
• Make a dilution series of the chosen model protein in the range:
0.1 mg/ml – 1.5 mg/ml (high protein range) OR
1 µg/ml – 25 µg/ml (low protein range)
• Mix the samples, standards and a blank (buffer, no protein) with BradfordUltra reagent in a microtiter plate:
For 0.1 mg/ml – 1.5 mg/ml protein (high range)
20 µl sample + 300 µl BradfordUltra reagent.
For 1 µg/ml – 25 µg/ml protein (low range)
150 µl sample + 150 µl BradfordUltra reagent
• Read absorbance immediately at 595 nm.
• Subtract the average 595 nm measurement for the blank from the 595 nm measurements of all other individual standards and unknown samples. Plot the average blank-corrected 595 nm measurement for each standard vs. concentration. Use the slope of this standard curve to estimate the protein concentration of the unknown samples.