Final 6

Question 1

What assay allows to quantify protein?

Answer 1

Bradford Assay

Question 2

What absorbance are protein quantified at? What amino acids absorb at this length?

Answer 2

Protein can be quantified at 280 nm
- Aromatic amino acids absorb at this length

Question 3

What is the range of concentrations should the protein be?

Answer 3

concentration should be 5ug – 200 ug concentration

Question 4

What is the principle of bradford assay?

Answer 4

Principle: Use a dye called commasie brilliant blue G – 20. The protein a dye bind and create a shift in absorbance.

Question 5

When is the dye added? What is the maximum absorption?

Answer 5

The dye is added the protein and absorbs at maximum absorption of 470nm (peak on OD)
Dye + Protein – Maximum absorption is 595nm (peak on OD)

Question 6

What is the spectrophotometer set to?

Answer 6

• Set spectrophotometer to 0-595

Question 7

What happens to absorbance when the protein binds to the dye? Are you looking for a curve? What value do you want?

Answer 7

• There is a shift in absorbance when the protein binds to the dye
• Not looking for a curve
• Just want OD value

Question 8

What is the chemistry of the Bradford Assay?

Answer 8

1. positively charged and basic amino acids bind to dye shifting the absorbance to a maximum of 595 nm

Question 9

What will not work for the bradford assay?

Answer 9

Bradford essay does not work on membrane proteins because the protein is between the lipids on the membrane
- Need to use a detergent where the hydrophobic molecule binds to the lipid part of the membrane. Detergent interferes with reading.
- Too acidic proteins will not work since the dye binds to the basic amino acids
- If using a quartz cuvette, the dye sticks to the wall of the cuvette and it interferes with the assay.

Question 10

What is the standard curve used for? What standard is used? Where is OD measured? What are the 3 main measurements we look at?

Answer 10

Standard curve needs to be used to determine the concentration of the protein
- Use BSA as a standard or immunoglobin protein.
Want OD at 595nm
Crude Extract + dye
Elution 1 + dye

Question 11

How is concentration determined for the protein of interest? What is the relationship of the OD and mg of the protein?

Answer 11

Plot the points and make the standard curve
Get the slope intercept equation.
Plug in the OD @ 595nm of your protein and get the concentration
OD and mg have a relationship. If you have OD of 7.5 then you know the amount
Take the number of ul, get the ug from the OD graph then convert it to 1ul
Ex: OD = 0.7, from graph and equation get ug=0.4, amount used = 5ul
0.4ug = 400ng
400ng/5ul= 80ng/1ul