The Bradford Protein Assay Quiz

Coomassie brilliant blue G-250

anionic (blue), neutral (green), and cationic (red)

Through van der Waals force and electrostatic interactions

It donates its free electron to the ionizable groups on the protein

One disadvantage of the Bradford assay is its linearity over a short range, typically from 0 µg/mL to 2000 µg/mL, often requiring dilutions of a sample before analysis. In making these dilutions, errors in one dilution can compound in further dilutions, resulting in a linear relationship that may not always be accurate.

Basic conditions and detergents, such as SDS, can interfere with the dye's ability to bind to the protein through its side chains, thereby affecting the accuracy of the assay.

The reagents in the Bradford assay tend to stain the test tubes, necessitating the use of separate test tubes for each sample. This can affect the absorbance reading and introduce variability into the analysis process.

The non-linearity in the Bradford assay is partly due to the equilibrium between two different forms of the dye, which is perturbed by adding the protein, leading to inaccurate results.

The addition of cyclodextrins to the assay mixture can alleviate the inhibition by detergents in the Bradford assay.

The Coomassie Blue G250 dye's preference to bind to arginine and lysine groups of proteins can result in a varied response of the assay between different proteins, introducing variability into the analysis process.

Increasing the pH by adding NaOH or adding more dye can correct the variation caused by the dye's preference for specific amino acids, but it results in a less sensitive assay and becomes sensitive to detergents.

To prepare a series of standards, dilute with 0.15 M NaCl to final concentrations of 0 (blank = No protein), 250, 500, 750, and 1500 µg/mL. Also prepare serial dilutions of the unknown sample to be measured. Add 100 µL of each to a separate test tube and then add 5.0 mL of Coomassie Blue to each tube and mix by vortex, or inversion.

The wavelength shift observed in the Bradford protein assay is from 465 nm to 595 nm.

The Bradford protein assay is less susceptible to interference by various chemical compounds like sodium, potassium, and carbohydrates.

High concentrations of buffer in the protein sample can lead to an overestimation of protein concentration in the Bradford protein assay.

The Bradford protein assay does not require measurement in the UV range and can avoid complications arising from the presence of aromatic amino acids and nucleic acids in the sample.

The absorbance is measured at 595 nm in the Bradford protein assay.

The Bradford protein assay can measure protein quantities as little as 1 to 20 $\mu$g.

The dye reagent can remain stable at room temperature for up to 2 weeks in the Bradford protein assay.

The Bradford protein assay is compatible with protein samples containing salts, solvents, buffers, preservatives, reducing agents, and metal chelating agents.

Protein concentration is determined by extrapolating using the slope of the line from the linear graph acquired in the Bradford protein assay.

The dye binds to the proteins through electrostatic and Van Der Waals interactions in the Bradford protein assay.

The Bradford protein assay can be performed using a spectrophotometer or a mobile smartphone camera (RGBradford method) for absorbance measurement at 595 nm.

The Bradford protein assay is cost-effective, easy to use, and has high sensitivity for protein, making it a favorable choice for protein quantification.