Protein Assays

Question 1

There are four main types of protein assays when it comes to quantification. What are they?

Answer 1

(1) Bradford
(2) Lowry
(3) BCA
(4) UV

Question 2

What type of dye does the Bradford assay use?

Answer 2

Coomassie Blue G-250

Question 3

To what does Coomassie Blue G-250 primarily bind?

Answer 3

Basic and aromatic amino acids in the polypeptide with a particular affinity to arginine

Question 4

Why can Bradford assay results vary slightly from one protein to the next?

Answer 4

Because Coomassie Blue G-250 has a particular affinity for basic and aromatic residues, especially arginine

Question 5

Under what conditions does an absorbance shift occur for Coomassie Blue?

Answer 5

Acidic

Question 6

What happens to the color of Coomassie Blue under acidic conditions and in the presence of proteins?

Answer 6

A shift from red to blue with blue corresponding to an increase in absorbance

Question 7

The Bradford assay is very good at negating most interference from other reagents in a sample, including ________________, but not if _______________ is present.

Answer 7

EDTA

SDS

Question 8

The __________________ assay is sensitive to bovine serum albumin.

Answer 8

Bradford

Question 9

Because of its sensitivity to bovine serum albumin, the Bradford assay may result in an _____________________ (under or overestimation) of protein concentration in a sample.

Answer 9

Underestimation

Question 10

Besides bovine serum albumin, what else can be used to generate a standard curve in a Bradford assay?

Answer 10

Immunoglobulin G

Question 11

What are the five advantages of the Bradford assay?

Answer 11

(1) Easy and simple procedure to follow
(2) Absorbance is measured at 595 nm in the visible range
(3) Relatively quick
(4) Extremely sensitive
(5) Leads to production of linear graph for easy extrapolation

Question 12

What’s a disadvantage of the Bradford assay?

Answer 12

Its linearity occurs over a short range (o ug/mL to 2,000 ug/mL), which requires sample dilutions

Question 13

The Lowry and BCA assays use the _____________ _______________ for determining protein concentration.

Answer 13

Biuret reaction

Question 14

In the ___________ ____________, copper sulfate in an alkaline solution reacts with peptide bonds in a protein, reducing copper II to copper I.

Answer 14

Biuret reaction

Question 15

What reacts with peptide bonds in a Biuret reaction?

Answer 15

Copper sulfate

Question 16

Under what conditions does a Biuret reaction occur?

Answer 16

Alkaline

Question 17

The ____________ assay occurs in two steps. The first step is the Biuret reaction. The second step is the _____________ of aromatic proteins.

Answer 17

Lowry

Oxidation

Question 18

In the second step of the Lowry assay, aromatic amino acids are oxidized via the reduction of ____________________ by a ___________________ ______________ _____________ from the preceding Biuret reaction.

Answer 18

FCR (Folin-Ciocalteau Reagent)

Copper-peptide bond complex

Question 19

The Lowry assay is sensitive and accurate for ___________ (small or large) peptides but can be time-consuming and is sensitive to interference.

Answer 19

Small

Question 20

The ___________________ _____________ assay also uses the Biuret reaction, reacting directly with copper to produce a purple product under alkaline conditions.

Answer 20

Bicinchoninic acid (BCA)

Question 21

What does BCA stand for?

Answer 21

Bicinchoninic acid

Question 22

In the Lowry assay, what two amino acid residues can also reduce FCR (Folin-Ciocalteau Reagent)?

Answer 22

Tyrosine

Tryptophan

Question 23

After the Biuret reaction, the copper I reacts directly with _________________ _____________ in the ______________ __________ assay to produce a stable purple product under alkaline conditions.

Answer 23

Bicinchoninic acid (BCA)
Bicinchoninic acid (BCA)

Question 24

UV light assays detect what types of amino acids?

Answer 24

Aromatic

Question 25

Although the UV light assay is very sensitive and fast, it requires _____________ protein samples.

Answer 25

Pure