The Bradford Protein Assay Quiz

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Questions and Answers

The Bradford protein assay is based on the absorbance shift of which dye?

Coomassie brilliant blue G-250

What are the three forms of the Coomassie brilliant blue G-250 dye?

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

How does the dye form a complex with the protein's carboxyl group and amino group?

Through van der Waals force and electrostatic interactions

What is the role of the red form of Coomassie dye in the formation of the complex with the protein?

It donates its free electron to the ionizable groups on the protein Signup and view all the answers

Explain one disadvantage of the Bradford assay and how it can affect the accuracy of the analysis.

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. Signup and view all the answers

How do basic conditions and detergents, such as SDS, interfere with the Bradford assay?

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. Signup and view all the answers

Explain the impact of the reagents in the Bradford assay staining the test tubes on the analysis process.

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. Signup and view all the answers

How does the equilibrium between two different forms of the dye contribute to the non-linearity of the Bradford assay?

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. Signup and view all the answers

What modification can be made to the Bradford assay to alleviate the inhibition by detergents?

The addition of cyclodextrins to the assay mixture can alleviate the inhibition by detergents in the Bradford assay. Signup and view all the answers

Explain the preference of the Coomassie Blue G250 dye to bind to arginine and lysine groups of proteins and its impact on the 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. Signup and view all the answers

What is the role of increasing the pH by adding NaOH or adding more dye in modifying the original Bradford assay?

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. Signup and view all the answers

Explain the procedure for preparing a series of standards and unknown samples for the Bradford assay.

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. Signup and view all the answers

What is the wavelength shift observed in the Bradford protein assay?

The wavelength shift observed in the Bradford protein assay is from 465 nm to 595 nm. Signup and view all the answers

What chemical compounds does the Bradford protein assay show less susceptibility to interference from?

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

What can lead to an overestimation of protein concentration in the Bradford protein assay?

High concentrations of buffer in the protein sample can lead to an overestimation of protein concentration in the Bradford protein assay. Signup and view all the answers

What are the advantages of the Bradford protein assay over other methods?

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. Signup and view all the answers

At what wavelength is the absorbance measured in the Bradford protein assay?

The absorbance is measured at 595 nm in the Bradford protein assay. Signup and view all the answers

What quantity of protein can the Bradford protein assay measure?

The Bradford protein assay can measure protein quantities as little as 1 to 20 $\mu$g. Signup and view all the answers

How long can the dye reagent remain stable at room temperature in the Bradford protein assay?

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

What types of protein samples is the Bradford protein assay compatible with?

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

How is protein concentration determined using the linear graph acquired from the Bradford protein assay?

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

Through what interactions does the dye bind to the proteins in the Bradford protein assay?

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

What are the two methods for absorbance measurement 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. Signup and view all the answers

What are some characteristics that make the Bradford protein assay a favorable choice for protein quantification?

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

Flashcards

What is the principle behind the Bradford protein assay?

The Bradford protein assay utilizes Coomassie brilliant blue G-250 dye to quantify protein concentration. The dye's color changes upon binding to proteins, shifting the absorption spectrum from 465 nm to 595 nm.

What are some factors that can influence the Bradford assay's accuracy?

The assay is relatively insensitive to the presence of common chemicals like sodium, potassium, and carbohydrates. However, high levels of detergents, especially SDS, can interfere with the results.

How can buffer concentration affect the Bradford assay?

The assay may overestimate protein concentration if the sample contains a high concentration of buffer. This is because the buffer's conjugate base can consume free protons in the solution.

What is an advantage of the Bradford protein assay over other methods?

The Bradford assay is beneficial because it does not require measurements in the UV range. This avoids complications from the presence of aromatic amino acids and nucleic acids in the sample.