9. Bradford Protein Assay: Chemistry, Calibration & Calculations

all answers r A rn, redo placement later, or remove MQ idk.

Which reagent produces the color response in the Bradford assay?

Coomassie Brilliant Blue G-250

At what wavelength did the report measure Bradford absorbance?

595 nm

What is the chemical basis of the Bradford color change?

Protein binding stabilizes the blue anionic form of the dye

Why did the report prepare BSA standards?

To create a calibration curve relating known protein concentration to absorbance

In the standard-curve equation, what does y represent?

Absorbance at 595 nm

Why should an unknown's absorbance fall within the standard curve range?

Interpolation within the calibrated range is more reliable than extrapolation

Why is a reagent/sample blank included?

To account for background absorbance from buffer and assay reagents

What is the best reason to dilute a concentrated unknown before measuring it?

To bring its absorbance into the calibrated linear range

The report's 1:100 unknown had an absorbance of 0.322 and a calculated diluted concentration of 0.463 mg/mL. What original concentration estimate follows?

46.3 mg/mL

Why was the 1:100 lactase dilution considered the most reliable estimate?

Its absorbance fell within the BSA calibration range

What does the Bradford concentration of crude lactase extract represent?

Total protein concentration in the extract

Which procedural error could increase variability in a Bradford assay?

Inconsistent pipetting or incomplete mixing

How would you teach standard-curve use to a novice?

Use known standards to draw the response line, then map the unknown absorbance back to concentration

Why must the dilution factor be applied after calculating the diluted sample concentration?

The curve gives the concentration in the measured diluted tube, not the original stock

Which comparison of Bradford, Biuret, and BCA matches the report appendix?

Bradford uses dye binding; Biuret forms a Cu(II)-peptide complex; BCA detects Cu(I)-BCA complex

A sample contains detergent. Which caution is specifically noted for Bradford assay?

Detergents can interfere with the dye-binding assay