Lab 3: Determination of Protein Concentrations

Question 1

What are the four spectrophotometric protein quantification assays?

Answer 1

Biuret, Lowry, Bradford (Dye Binding) and UV spectrophotometry

Question 2

What is the standard curve used for? What are the axis?

Answer 2

Quantification of protein in a sample for a protein assay

- plot of absorbance (Y axis) vs varying amount of known substances (X axis)

Question 3

What does the linear region of a standard curve represent?

Answer 3

Where absorbance is directly proportional to amount of protein in the sample (middle of standard curve)

Question 4

What is the coefficient of determination?

Answer 4

R^2 value, found on standard curve, how well data points are in linear relationship

Question 5

What do sensitivity and precision mean?

Answer 5

Sensitivity: How small amount of sample is needed for the test
Precision: reproducibility (how close to the expected)

Question 6

For the Biuret, what are the proteins mixed with and how does it work? How is it described in terms of quantification, reliability and sensitivity?

Answer 6

• proteins mixed with alkaline solution of copper salt
• proteins with 2+ peptide bonds chelate cupric ions (Cu2+) in alkaline solution making purple complex
• it is quantitative, reliable but insensitive (needs mg of proteins)

Question 7

What is the range that the Biuret Assay is used at?

Answer 7

540 to 560nm

Question 8

For the Lowry, what are the proteins mixed with and how does it work? How is it described in terms of quantification, reliability and sensitivity?

Answer 8

2-step reaction: (modified Biuret)

• complexation of peptide bonds with cupric ions in alkali (Biuret reaction)
• then the reduction of Folin-Ciocalteu reagent by Biuret complexes, and tyrosine, tryptophan and cysteine and histidine
• makes blue colour and more sensitive than the Biuret

Question 9

What range is the Lowry assay absorbed at?

Answer 9

750 nm (red region)

Question 10

For the Bradford, what are the proteins mixed with and how does it work? How is it described in terms of quantification, reliability and sensitivity?

Answer 10

• mixed with Coomassie Brilliant Blue dye
• acidic pH dye binds arginine, histidine, phenylalanine, tryptophan, and tyrosine residues
• shift in absorbance from 465nm to 595 nm due to stabilization of the anionic dye
• binding happens in 5 minutes and stable for 1 hour

Question 11

What is the thing that binds to the proteins in the Bradford assay?

Answer 11

Coomassie Brilliant Blue G is a protein binding dye

Question 12

What is the range of absorbance for Bradford?

Answer 12

465 to 595nm (becomes more stable so shifts)