Week 2 Day 1 Measuring Protein Concentration handout

The Bradford assay was developed by __________ in 1976.

Dr. Marion Bradford

The dye used in the Bradford protein assay is called __________.

Coomassie Brilliant Blue or Coomassie G-250

The shift in maximum light absorbance when protein is present in the Bradford assay is from __________ nm to __________ nm.

465 nm to 595 nm

To create a standard curve in the Bradford protein assay, known concentrations of __________ are used.

protein

If the R2 value obtained from the standard curve is above __________, the curve is considered usable.

0.90

Bovine serum albumin (BSA) is used as a __________ in the Bradford assay.

protein standard

To protect proteins from degradation, samples should be kept on __________.

ice

One of the precautions to take when working with protein samples is to __________ the bench surfaces with 70% ethanol.

wipe down

During the Bradford assay, the __________ is used to measure light absorbance at 595 nm.

spectrophotometer

When performing the Bradford assay, the __________ is the independent variable, while absorbance is the dependent variable.

concentration

Why is the Bradford assay important in protein analysis?

The Bradford assay is important because it provides a quick, sensitive, and reliable method to quantify protein concentrations in solution.

How does the presence of protein affect the absorbance in the Bradford assay?

The presence of protein causes a shift in the absorbance peak from 465 nm to 595 nm, which indicates the binding of the dye to the protein.

What factors could affect the accuracy of the Bradford assay results?

Factors that could affect accuracy include the presence of contaminants, the choice of protein standard, and the quality of the reagents used.

Discuss how you would prepare a standard curve for the Bradford assay and its significance.

To prepare a standard curve, known concentrations of a protein standard, such as BSA, are measured for absorbance at 595 nm to create a linear relationship between known concentration and absorbance, which is crucial for determining the concentration of unknown samples.

Explain the significance of using BSA as a protein standard in the Bradford assay.

BSA is commonly used as a standard because it is well-characterized, provides consistent results, and its absorbance properties are similar to many other proteins, facilitating accurate comparisons.