Quantitative of protein
Huda Hania
Methods of Quantitative of protein
• Method 1: protein assay based on dye binding assay
• Method 2: protein assay based on alkaline copper
Method 1: protein assay based on dye binding assay
• BRADFORD METHOD
• Use of coomassie G-250 dye in a colorimetric reagent
for the detection and quantitation of total protein .
• In the acidic environment of the reagent, protein binds to
the coomassie dye.
• This results in a spectral shift from the reddish/brown
form of the dye (absorbance maximum at 465nm) to the
blue form of the dye (absorbance maximum at 610nm).
• The difference between the two forms of the dye is
greatest at 595nm, so that is the optimal wavelength to
measure the blue color from the coomassie dye-protein
complex
• Development
of
color
in
coomassie
dye-based
(Bradford) protein assays has been associated with the
presence of certain basic amino acids (primarily arginine,
lysine and histidine) in the protein.
• Free amino acids, peptides and low molecular weight
proteins do not produce color with coomassie dye
reagents. (unbound) forms are green or red.
Samples treated with the Bradford
assay. The brown sample (lower
absorbance) contains no protein, while
the blue sample (higher absorbance)
contains protein.
The amount of protein in the second
sample can be determined by
comparison to a standard curve
• Advantages: This assay is quick, and the reagent is not
affected by the presence of reducing agents, like DTT
• Disadvantages: Basic conditions and detergents, such
as SDS, can interfere with the dye’s ability to bind to the
protein
Biuret Test
• Principle:
• Under alkaline conditions substances containing two or
more peptide bonds form a purple complex with copper
salts in the reagent.
•
• Biuret Reagent contains:
•
Hydrated Copper sulphate – this provides the
Cu (II) ions which form the chelate complex.
Cu (II) ions give the reagent its characteristic
blue color.
• Potassium hydroxide does not participate in the
reaction but provides the alkaline medium.
• Potassium sodium tartrate (KNaC4H4O6·4H2O)
stabilizes the chelate complex
• The Biuret reaction can be used to measure the
concentration of proteins because peptide bonds
occur with the same frequency per amino acid in
the peptide.
• The intensity of the color, and hence the
absorption at 540 nm, is directly proportional to
the protein concentration, according to the
Beer-Lambert law.
tubes
D.W
Protein
standard
1
0.5
0
0
2
0.4
0.1
0.4
3
0.3
0.2
0.8
4
0.2
0.3
1.2
5
0.1
0.4
1.6
6
0
0.5
2
Unknown
0
0
1.
2.
3.
4.
Unknown
0.5
Add 1ml of biuret reagent to each tube and mix
Incubation 30 min
Read at 540 nm
Draw the standard curve and measure the unknown
Conc
• A standard curve is a type of graph used as a quantitative research
technique. Multiple samples with known properties are measured
and graphed, which then allows the same properties to be
determined for unknown samples by interpolation on the graph. The
samples with known properties are the standards, and the graph is
the standard curve.
•
For example, a standard curve for protein concentration is often
created using known concentrations of bovine serum
Reference range
• Reference range for total proteins is 66.6 to 81.4 g/L