Protein Assays Flashcards
There are four main types of protein assays when it comes to quantification. What are they?
(1) Bradford
(2) Lowry
(3) BCA
(4) UV
What type of dye does the Bradford assay use?
Coomassie Blue G-250
To what does Coomassie Blue G-250 primarily bind?
Basic and aromatic amino acids in the polypeptide with a particular affinity to arginine
Why can Bradford assay results vary slightly from one protein to the next?
Because Coomassie Blue G-250 has a particular affinity for basic and aromatic residues, especially arginine
Under what conditions does an absorbance shift occur for Coomassie Blue?
Acidic
What happens to the color of Coomassie Blue under acidic conditions and in the presence of proteins?
A shift from red to blue with blue corresponding to an increase in absorbance
The Bradford assay is very good at negating most interference from other reagents in a sample, including ________________, but not if _______________ is present.
EDTA
SDS
The __________________ assay is sensitive to bovine serum albumin.
Bradford
Because of its sensitivity to bovine serum albumin, the Bradford assay may result in an _____________________ (under or overestimation) of protein concentration in a sample.
Underestimation
Besides bovine serum albumin, what else can be used to generate a standard curve in a Bradford assay?
Immunoglobulin G
What are the five advantages of the Bradford assay?
(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
What’s a disadvantage of the Bradford assay?
Its linearity occurs over a short range (o ug/mL to 2,000 ug/mL), which requires sample dilutions
The Lowry and BCA assays use the _____________ _______________ for determining protein concentration.
Biuret reaction
In the ___________ ____________, copper sulfate in an alkaline solution reacts with peptide bonds in a protein, reducing copper II to copper I.
Biuret reaction
What reacts with peptide bonds in a Biuret reaction?
Copper sulfate
Under what conditions does a Biuret reaction occur?
Alkaline
The ____________ assay occurs in two steps. The first step is the Biuret reaction. The second step is the _____________ of aromatic proteins.
Lowry
Oxidation
In the second step of the Lowry assay, aromatic amino acids are oxidized via the reduction of ____________________ by a ___________________ ______________ _____________ from the preceding Biuret reaction.
FCR (Folin-Ciocalteau Reagent)
Copper-peptide bond complex
The Lowry assay is sensitive and accurate for ___________ (small or large) peptides but can be time-consuming and is sensitive to interference.
Small
The ___________________ _____________ assay also uses the Biuret reaction, reacting directly with copper to produce a purple product under alkaline conditions.
Bicinchoninic acid (BCA)
What does BCA stand for?
Bicinchoninic acid
In the Lowry assay, what two amino acid residues can also reduce FCR (Folin-Ciocalteau Reagent)?
Tyrosine
Tryptophan
After the Biuret reaction, the copper I reacts directly with _________________ _____________ in the ______________ __________ assay to produce a stable purple product under alkaline conditions.
Bicinchoninic acid (BCA) Bicinchoninic acid (BCA)
UV light assays detect what types of amino acids?
Aromatic
Although the UV light assay is very sensitive and fast, it requires _____________ protein samples.
Pure
