Protein Estimation by Biuret Method in Biochemistry Practical
1
B
C
H
3
0
2
[
P
r
a
c
t
i
c
a
l
]
•
Quantitative assays
Quantitative assays
d
etermine the
concentration
of a substance.
•
Protein assays are one of the most widely used methods in life science research.
•
Estimation of protein concentration is necessary in protein purification, cell
biology, molecular biology and other research applications.
•
Is necessary before processing protein samples for isolation, separation and
analysis.
2
3
Specificity
of an assay relates to how
good the assay is in discriminating
between the requested analyte and
interfering substances.
Sensitivity
of an assay is a measure
of how little of the analyte the
method can detect.
4
•
There are a wide variety of protein assays available, but each assay has its own
advantages
and
limitations
.
•
The factors that you should consider in choosing a method:
Sensitivity.
The presence of interfering substance.
Time available of the assay.
•
It is a graph that shows the relationship between different
known concentrations
of a
substance and the
absorbance at a specific wave length
.
•
Standard curve are most commonly used to determine the
concentration of a unknown
substance
, using serial dilution of solutions of known concentrations[
standard solution
],
such as (Bovine serum Albumin) BSA and casein
.
•
There is
a linear relationship
between
absorbance
absorbance
and
concentration.
concentration.
5
6
7
8
•
Determination of an unknown concentration for protein sample by Biuret
method.
•
Getting familiar with standard curve.
Principle:
•
In this reaction, proteins form a
purple colored complex
with CuSO
4
(copper
sulfate) in a strongly
alkaline solution.
•
When
peptide bonds
in proteins and peptides treated with an
alkaline solution
of
dilute copper sulfate (Biuret reagent ) a
violet
color is formed
A
positive test
is
indicated by the formation of a
violet
color.
•
The
color
density is
proportional
to amount of
proteins
present.
9
10
There is
a linear relationship
between
purple colour
purple colour
developed
developed
and
concentration.
concentration.
From lower to higher concentration
From lower to higher concentration
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12
Method:
•
Set up 8 tubes as follows:
•
Let the tubes stand at room temperature for 10 min.
•
Read absorbance at 540 nm against the blank.
13
Results:
•
Plot a standard curve for absorbance at 540 nm against Albumine std. concentration (mg/dl).
•
From the standard curve find out the unknown protein concentration.
Protein estimation plays a vital role in various research applications. This practical guide covers the importance of determining protein concentration, specificity, sensitivity of methods, familiar spectrophotometric methods, standard curves, and determining unknown concentrations using Biuret method. The content includes informative visuals to aid in understanding the concepts and objectives of the practical.
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Presentation Transcript
Quantitative Proteins Estimation by Biuret method BCH302 [Practical] 1
Importance of determining concentration of protein : Quantitative assays determine the concentration of a substance. Protein assays are one of the most widely used methods in life science research. Estimation of protein concentration is necessary in protein purification, cell biology, molecular biology and other research applications. Is necessary before processing protein samples for isolation, separation and analysis. 2
Specificity and Sensitivity of a method: Sensitivity of an assay is a measure of how little of the analyte the method can detect. Specificity of an assay relates to how good the assay is in discriminating between the requested analyte and interfering substances. 3
Most familiar spectrophotometric methods for Determination of proteins concentration : There are a wide variety of protein assays available, but each assay has its own advantages and limitations. Warburg Christian (A280/A260) Bicinchoninic acid method Biuret Lowry Bradford The factors that you should consider in choosing a method: Sensitivity. The presence of interfering substance. Time available of the assay. 4
Standard curve: It is a graph that shows the relationship between different known concentrations of a substance and the absorbance at a specific wave length. Standard curve are most commonly used to determine the concentration of a unknown substance, using serial dilution of solutions of known concentrations[standard solution], such as (Bovine serum Albumin) BSA and casein. There is a linear relationship between absorbance and concentration. 5
Determination of unknown concentration by standard curve: 6
Objectives: Determination of an unknown concentration for protein sample by Biuret method. Getting familiar with standard curve. 8
Biuret method: Principle: In this reaction, proteins form a purple colored complex with CuSO4 (copper sulfate) in a strongly alkaline solution. When peptide bonds in proteins and peptides treated with an alkaline solution of dilute copper sulfate (Biuret reagent ) a violet color is formed A positive test is indicated by the formation of a violet color. The color density is proportional to amount of proteins present. 9
From lower to higher concentration There is a linear relationship between purple colour developed and concentration. 10
Experiment 1 : Estimation of protein concentration by Biuret method Method: Set up 8 tubes as follows: Tube Water (ml) Bovine serum albumin Standard Concentration [5g/l] - sample [unknown concentration] Biuret reagent( ml ) Blank 2 - 1 1.6 0.4 - 2 1.2 0.8 - 3 1 1 - 4 0.8 1.2 - 3 5 0.6 1.4 - 6 0.4 1.6 - Unknown concentration sample - - 2 Let the tubes stand at room temperature for 10 min. Read absorbance at 540 nm against the blank. 12
Experiment 1 : Estimation of protein concentration by Biuret method Results: Tube Albumin concentration (mg/dl) [X- axis] Absorbance at 540nm [Y-axis] 1 C1 X V1 = C2 X V2 500 x 0.4 = ? x 2 2 3 4 5 6 Plot a standard curve for absorbance at 540 nm against Albumine std. concentration (mg/dl). From the standard curve find out the unknown protein concentration. 13
