Lab2: Determination of Protein Concentration

Question 1

Beer’s law ?

Answer 1

the intensity of a beam of monochromatic light ( light composed of a single wavelength) passiong through a solution containing chromophoric (light absorbing) molecules or ions will be deminished. Some of the light energy is absorbed by the molecules and the transmitted light is less intense than the incident beam.

Question 2

Lambert’s law?

Answer 2

• the amount of light that is absorbed by a solution keeping all conditions constant is directly proportional to the thickness of the layer through which the light must travel.

Question 3

Absorbance formula?(beer)

Answer 3

= intensity of transmitted/intensity of incident beam

Question 4

Absorbance formula? (lambert?)

Answer 4

E x thickness of solution layer

Question 5

Which method is the least sensitive?

Answer 5

Biuret!

Question 6

Explain the biuret method!

Answer 6

• two peptide bonds are required for the complex so only tripeptides and larger are detected in this test. The complex formation and resulting intensity of the colour are reproducible for a protein and similar absorption spectra have been observed for different proteins making the test suitable as a total protein determination.
• BSA is used or lysozyme for standards for calibration as they can be obtained pure and give reproducible complex formation

Question 7

Purpose of a blank tube?

Answer 7

to set the zero absorbance point and because the negatives and positive interfere with the reagents.
L> it contains everything but the compound of interest so any absorbance measured thereafter will be due to the addition of the compound!

Question 8

Advantages of the biuret assay?

Answer 8

colour produced is stable for 1-2 hours which is longer than the lowry method. The colour intensity and complex formation is reproducible and similar absorbance. Similarly absorbance ranges are seen across many proteins therefore it is a good total protein determination test.
- fewer possible interferences

Question 9

Disadvantages of the biuret assay?

Answer 9

cannot detect tripeptides or larger so it cannot accurately measure small proteins. It is not a very sensitive test. Interferenes from tris aminomethone, free histidine or excess ammonium ions can make the test unreliable.

Question 10

Why does the biuret reagent have sodium potassium tartrate in it?

Answer 10

• stabilizes copper ions

Question 11

Why is BSA not used in the lowry test?

Answer 11

it contains low concentrations of tyrosine and tryptophan which leads to a low response in this test.

Question 12

In the biuret test which part of the protein reacts with the reagent producing colour?

Answer 12

• dilute alkaline copper sulfate solution treatment produces a pink or purple complex between the Cu 2+ ion and the nitrogen atoms of the protein.

Question 13

In the lowry test which part of the protein reacts with the reagent producing colour?

Answer 13

• biuret complex is produced by alkaline copper and a second blue green colour forms by the reaction of FC phenol with the tyrosine and tryptophan residues in peptides or proteins.

Question 14

In the biuret test which part of the protein reacts with the reagent producing colour?

Answer 14

1 to 20 mg/mL

Question 15

In the lowry test which part of the protein reacts with the reagent producing colour?

Answer 15

5 to 300 ug/mL
L> makes it perfect for following protein purification where sample sizes are limited.
** much more sensitive

Question 16

On your table when you have the absorbances found…but need mg protein per tube…calc?

Answer 16

you multiple Lysozyme xmg/mL by the amount of protein present in each tube

Question 17

How do you calculate the ratio of the concentrations found for each unknown solution with the ratio of method of sensitivity?

Answer 17

Ratio of unknown = B/L ( avg unknown from B/ avg unknown from L)
Ratio of sensitivity of method= B/L
= 20mg/mL-2mg/mL / 0.300mg/mL-0.05mg/mL
ratio of the two…just compare them!
- order of magnitude statement is needed !