BradfordUltra – Detergent compatible protein quantitation
When Coomassie dye binds protein in an acidic medium, an immediate shift in absorption maximum occurs from 465 nm to 595 nm with a concomitant color change from brown to blue. Protein concentrations are estimated by reference to absorbances obtained for a series of standard protein dilutions, which are assayed alongside the samples with unknown protein concentrations.
Expedeon’s BradfordUltra is an improvement over classical Bradford formulations which cannot tolerate detergent contamination of the protein samples. In addition, the BradfordUltra reagent shows excellent linearity for a defined range of protein concentrations and shows significantly less protein-to-protein variation than is observed with other Bradford-type Coomassie formulations.
Comparison of Expedeon’s BradfordUltra Assay with Pierce’s Coomassie Protein Assay. The graph shows the average blank corrected A595 measurements for detergents samples (no protein). When detergents are present in the sample, competitor Bradford assay solutions give high blank readings making accurate detection of the protein sample impossible. BradfordUltra is not affected by the detergents compared to Classic formulations and provides excellent signal to noise ratios and accurate determintation of the protein concentration regardless of the detergent presence.
Standard curves obtained with BradfordUltra are unaffected by the presence of detergents.
Standard curves obtained with classical Bradford formulation are significantly affected by thepresence of detergents resulting in loss of sensitivity and inaccurate results.
1) 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 ug/ml – 25 ug/ml (low protein range)
2) Mix the samples, standards and a blank (buffer, no protein) with BradfordUltra reagent.
High Protein Range: 1 part sample for 15 parts reagent
Low Protein Range: 1 part sample for 1 part reagent
3) Read absorbance at 595 nm.
4) Calculate concentration:
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.