HiPer® Protein Estimation Teaching Kit
(Quantitative)
Product Code: HTBC005
Number of experiments that can be performed: 5
Duration of Experiment
Protocol
Biuret assay:1 hour
Folin – Lowry Assay: 2 hours
Bradford assay: 1 hour
Storage Instructions:
The kit is stable for 6 months from the date of receipt
Store all the kit contents as specified in the brochure
1
Index
Sr. No.
Contents
Page No.
1
Aim
3
2
Introduction
3
3
Materials Required But Not Provided
3
4
Storage
3
5
Biuret Assay
3
6
Folin – Ciocalteau (Lowry) Assay
7
6
Principle
Kit contents
Important Instructions
Procedure
Observation and Result
Interpretation
Dye Binding (Bradford) Assay
8
Principle
Kit contents
Important Instructions
Procedure
Observation and Result
Interpretation
9
Principle
Kit contents
Important Instructions
Procedure
Observation and Result
Interpretation
Troubleshooting Guide
12
2
Aim:
To determine the concentration of a protein by three commonly used methods:
1. Biuret Assay
2. Folin – Ciocalteau (Lowry) Assay
3. Dye Binding (Bradford) Assay
Introduction:
HiPer® Protein Estimation Teaching Kit is designed for rapid and accurate determination of proteins by three most
commonly used techniques, each having features that suit it to a particular use. It enables the determination of the
concentration of proteins which is frequently required in biochemical work. There is no completely satisfactory
single method to determine the concentration of protein in any given sample. Most protein assays take advantage of
a reaction between a reagent dye and the protein of interest that will shift or increase the absorbance of a particular
wavelength. The choice of the method depends on the nature of the protein, the nature of the other components in
the protein sample, desired speed, accuracy and sensitivity of the assay.
Materials Required But Not Provided:
Glass wares: 1 ml and 10 ml Pipettes, cuvettes, test tubes
Reagents: Distilled Water
Other requirements: Spectrophotometer/Colorimeter to determine the absorbance in given range, Micropipette and
tips, test tube stand
Storage:
HiPer® Protein Estimation Teaching Kit can be stored at 2-8oC for up to 6 months without showing any reduction in
performance. Read Important Instructions before starting the experiment. Store all the reagents as specified in the
brochure.
1. Biuret Assay for Protein Estimation:
Principle:
The Biuret is a general protein assay for batches of material for which yield is not a problem. In Biuret reaction
peptides containing three or more amino acid residues form a colored chelate complex with cupric ions (Cu2+) in an
alkaline environment containing sodium potassium tartrate. This became known as the biuret reaction because it is
chemically similar a complex that forms with the organic compound biuret (NH2-CO-NH-CO-NH2) and the cupric
ion. Biuret, a product of excess urea and heat, reacts with copper to form a light blue tetradentate complex.
Fig 1: Biuret Reaction
3
Single amino acids and dipeptides do not give the biuret reaction, but tripeptides and larger polypeptides or proteins
will react to produce the light blue to violet complex that absorbs light at 540 nm. One cupric ion forms a colored
coordination complex with four to six nearby peptide bonds. The intensity of the color produced is proportional to
the number of peptide bonds participating in the reaction. Thus, the biuret reaction is the basis for a simple and
rapid colorimetric reagent of the same name for quantitatively determining total protein concentration.
Kit Contents:
Table 1: Enlists the materials provided in this kit for Biuret assay with their quantity and recommended storage
Quantity
Sr. No.
Product Code
Materials Provided
Storage
5 expts
4 ml
-200C
Biuret Reagent
110 ml
2-80C
TKC228
Protein Sample 1
1.2 ml
-200C
TKC229
Protein Sample 2
1.2 ml
-200C
1
TKC226
Protein Standard (50 mg/ml)
2
TKC227
3
4
Important Instructions:
1.
Read the entire procedure carefully before starting the experiment.
2.
All glasswares must be clean and protein free, otherwise it will interfere with the assay.
3.
The unknown and standard samples should be treated identically for accurate results.
4.
The assay should be carried out at the same time and in the same buffer conditions.
5.
Protein test samples provided are of different concentrations. (Protein Sample 1 and 2)
Procedure:
1.
Take nine tubes and label them as Blank and 1 to 8.
2.
Make dilutions of Protein (BSA) standards with concentrations of 10, 8, 6, 4, 2, 1 mg/200 ul by transferring
respective amount of BSA from the standard protein solution (50 mg/ml) and adjusting it to a total volume
of 200 µl by adding distilled water as mentioned in table 2.
3.
Add 2 ml of Biuret reagent to each test tube including the Blank and Unknown tubes. Mix well.
4.
Keep at room temperature for 10 minutes.
5.
Switch on the Spectrophotometer, select the wavelength at 540 nm and let it warm before taking the
absorbance (OD). First take the OD of Blank and make it zero.
4
6.
Remove Blank tube and take the OD of all the tubes and record it. Wash the cuvette after taking OD of each
sample.
Table 2:
Tube No.
Conc. of BSA
(mg)
Amt of Stock
(µl)
Amt of
Distilled Water
(µl)
Amt of Biuret
reagent (ml)
Blank
1
2
3
4
5
6
0.0
1
2
4
6
8
10
0.0
20
40
80
120
160
200
200
180
160
120
80
40
0.0
2
2
2
2
2
2
2
7
Test sample
1
8
Test Sample
2
200 µl
200 µl
2
2
Keep at room temperature for 10 minutes
Absorbance at
540 nm
7.
Plot a Standard Curve of absorbance at 540 nm on “Y” axis versus concentration of protein mg/200 µl
on “X” axis.
8.
Record the value “x” of Unknown from graph corresponding to the optical density reading of the test
sample.
Deatermination of Protein Concentration in Unknown Sample:
Protein concentration can be calculated using following formula:
Concentration of Unknown in “mg”
Protein Concentration in Test Sample = -------------------------------------------- x 1000 mg/ml
Volume of sample in “µl”
Observation and Result:
1
2
3
Fig 2: In Biuret Protein Assay the intensity of the colour increases with increasing protein concentration
5
Absorbance at 540 nm
Standard Curve for Protein Estimation by Biuret
Method
0.8
0.6
0.4
0.2
0
0
2
4
6
8
10
12
Protein Concentration m g/200µl
Interpretation:
The Biuret method is carried out by preparing a set of solutions with known protein concentrations and mixing them
with the Biuret reagent. A standard curve can be made and the concentrations of unknown protein sample can be
derived from the standard curve
2. Folin – Ciocalteau (Lowry) Assay for Protein Estimation:
Principle:
The Lowry’s method utilizes phenol reagent of Folin and Ciocalteau. This is essentially phosphotungstic
phosphomolybdic acid which can be reduced by phenols and many other substances with phenolic rings to
‘molybdenum blue’. Proteins reduce phenol reagent, which may be used therefore for their determination. However,
the amount of colour varies greatly with different proteins because it is entirely proportional to their content of
tyrosine and tryptophan, other amino acids having little effect. Pretreatment of proteins with alkali and a trace of
copper salt greatly increases the colour that is absorbed maximally at 750 nm.
Fig 3: Folin Lowry Reaction
6
Kit Contents:
Table 3: Enlists the materials provided in this kit for Lowry assay with their quantity and recommended storage
Quantity
Sr. No.
Product Code
Materials Provided
1
TKC230
Protein Standard (1mg/ml)
2
3
TKC231
TKC232
Folin’s Reagent (2N)
Solution A
4
TKC233
Solution B
5
6
7
TKC329
TKC234
TKC235
Solution C
Protein Sample 1
Protein Sample 2
5 expts
Storage
4 ml
-200C
15 ml
180 ml
RT
RT
2 ml
RT
2 ml
1.2 ml
1.2 ml
RT
-200C
-200C
Important Instructions:
1.
Read the entire procedure carefully before starting the experiment.
2.
All glasswares must be clean and protein free, otherwise it will interfere with the assay.
3.
The unknown and standard samples should be treated identically for accurate results.
4.
The assay should be carried out at the same time and in the same buffer conditions.
5.
Protein test samples provided are of different concentrations. (Protein Sample 1 and 2)
6.
Preparation of 1N Folin’s Reagent (5ml): Dilute Folin’s reagent (2N) with equal amount of Distilled water
on the day of use. ( 2.5 ml of Folin’s reagent + 2.5 ml of Distilled water)
7. Preparation of Alkaline Copper Reagent (30 ml): Mix 29.4 ml of Solution A with 0.3 ml of Solution B and
0.3 ml of Solution C on the day of use.
Procedure:
1.
Take ten tubes and label them as Blank and 1 to 9.
2.
Make dilutions of Protein (BSA) standards with concentrations of 200, 160, 120, 80, 40, 20, 10 µg/200 ul
by transferring respective amount of BSA from the standard protein solution (1 mg/ml) and adjusting it to a
total volume of 200 µl by adding distilled water as mentioned in table 4.
3.
Add 3 ml of Alkaline Copper reagent to each test tube including the Blank and Unknown tubes. Mix well.
4.
Keep at room temperature for 10 minutes.
5.
Add 0.5 ml of 1N Folin’s reagent to each test tube. Vortex the tubes and keep in Boiling Water Bath for 10
minutes
6.
Switch on the Spectrophotometer, select the wavelength at 750 nm and let it warm before taking the
absorbance (OD). First take the OD of Blank and make it zero
7
7.
Remove Blank tube and take the OD of all the tubes and record it. Wash the cuvette after taking OD of each
sample.
Table 4:
Tube No.
Conc. of BSA
(µg)
Amt of Stock
(µl)
Amt of
diluent (µl)
Alkaline
Copper
Reagent
Blank
1
2
3
4
5
6
7
8
Test
sample 1
9
Test
Sample 2
0.0
10
20
40
80
120
160
200
0.0
10
20
40
80
120
160
200
200
190
180
160
120
80
40
0.0
200 µl
200 µl
3
3
3
3
3
3
3
3
3
3
0.5
0.5
0.5
Keep at Room temperature for 10 minutes
Amt of
Folin’s
Reagent (ml)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Vortex and keep in Boiling Water Bath for 10 minutes
Absorbance
at 750 nm
8.
Plot a Standard Curve of absorbance at 750 nm on “Y” axis versus concentration of protein µg/200 µl
on “X” axis.
9.
Record the value “x” of Unknown from graph corresponding to the optical density reading of the test
sample.
Deatermination of Protein Concentration in Unknown Sample:
Protein concentration can be calculated using following formula:
Protein Concentration in Test Sample =
Concentration of Unknown in “µg”
------------------------------------------ x 1000 µg/ml
Volume of sample in “µl”
Observation and Result:
1
2
3
Fig 4: In Folin Lowry Protein Assay the intensity of the colour increases with increase in protein concentration
8
Absorbance at 750
nm
Standard Curve for Protein Estimation by Folin
Lowry Method
0.6
0.5
0.4
0.3
0.2
0.1
0
0
50
100
150
200
250
Protein Concentration µg/200 µl
Interpretation:
The Lowry’s method of protein estimation is carried out by preparing a set of solutions with known protein
concentrations and mixing them with the Folin’s reagent. A standard curve can be made and the concentrations of
unknown protein sample can be derived from the standard curve.
3. Bradford Assay for Protein Estimation:
Principle:
Bradford Assay for protein estimation is a rapid, simple and sensitive method for estimation. The dye used in this
method, Coomassie brilliant Blue G – 250 has an absorbance maximum of 465 nm in unbound state. When it
interacts with proteins, the dye turns blue in colour and the complex formed has a λ max of 595 nm. Thus proteins
can be estimated at 595 nm and the coloured complex formed is stable for one hour.
Fig 5: Bradford reaction
9
Kit Contents:
Table 5: Enlists the materials provided in this kit for Bradford assay with their quantity and recommended storage
Sr. No.
Product Code
Quantity
Materials Provided
Storage
5 expts
1
TKC230
Protein Standard (1 mg/ml)
0.4 ml
-200C
2
TKC236
Bradford’s Reagent
45 ml
2- 80C
3
TKC237
Protein Sample 1
1.2 ml
-200C
4
TKC238
Protein Sample 2
1.2 ml
-200C
Important Instructions:
1.
Read the entire procedure carefully before starting the experiment.
2.
All glasswares must be clean and protein free, otherwise it will interfere with the assay.
3.
The unknown and standard samples should be treated identically for accurate results.
4.
The assay should be carried out at the same time and in the same buffer conditions.
5.
Protein test Samples provided are of different concentrations. (Protein Sample 1 and 2)
Procedure:
1.
Take eight tubes and label them as Blank and 1 to 7.
2.
Make dilutions of Protein (BSA) standards with concentrations of 20, 16, 12, 8, 4 µg/200 µl by transferring
respective amount of BSA from the standard protein solution (1mg/ml) and adjusting it to a total volume of
200 µl by adding distilled water as mentioned in table 6.
3.
Add 1ml of Bradford’s reagent to each test tube including the Blank and Unknown tubes. Mix the contents
of each tube thoroughly by vortexing the tubes and incubate at RT for 10 minutes.
4.
Switch on the Spectrophotometer, select the wavelength at 595 nm and let it warm before taking the
absorbance (OD). First take the OD of Blank and make it zero.
5.
Remove Blank tube and take the OD of all the tubes within one hour and record it. Wash the cuvette after
taking OD of each sample.
10
Table 6:
Tube No.
Conc. of BSA
(µg)
Amt of Stock
(µl)
Amt of diluent
(µl)
Amt of Bradford
Reagent (ml)
Blank
1
2
3
4
5
0.0
4
8
12
16
20
0.0
4
8
12
16
20
200
196
192
188
184
180
1
1
1
1
1
1
6
Test sample
1
7
Test Sample
2
200 µl
200 µl
1
1
Vortex the tubes and incubate at RT for 10 minutes
Absorbance at
595 nm
6.
Plot a Standard Curve of absorbance at 595 nm on “Y” axis versus concentration of protein µg/200 µl
on “X” axis.
7.
Record the value “x” of Unknown from graph corresponding to the optical density reading of the
test sample.
Deatermination of Protein Concentration in Unknown Sample:
Protein concentration can be calculated using following formula:
Protein Concentration in Test Sample =
Concentration of Unknown in “µg”
-------------------------------------------- x 1000 µg/ml
Volume of sample in “µl”
Observation and Result:
1
2
3
Fig 6: In Bradford Assay the intensity of colour increases with increasing protein concentration
11
Absorbance at 595 nm
Standard Curve for Protein Estimation by
Bradford Method
0.8
0.6
0.4
0.2
0
0
5
10
15
20
25
Protein Concentration µg/200 µl
Interpretation:
The Bradford’s method of protein Estimation is carried out by preparing a set of solutions with known protein
concentrations and mixing them with the Bradford reagent. A standard curve can be made and the concentrations of
unknown protein sample can be derived from the standard curve.
Trouble shooting Guide:
Sr.No.
1.
Problem
Standards and Samples give
lower OD values than expected
although the Blank is ok
Possible Cause
Solution
Biuret, Folin’s and Bradford’s
reagents were not stored properly
Always store reagents as
mentioned in the brochure
Samples were cold while
performing the reaction
Allow samples to come to RT
before starting the reaction
Dye Precipitation was seen in
Bradford’s reagent
Filter the reagent before use
Absorbance was not measured at
correct wavelength
Measure absorbance at correct
wavelength as mentioned in
the brochure
Technical Assistance:
At HiMedia we pride ourselves on the quality and availability of our technical support. For any kind of Technical
assistance mail at mb@himedialabs.com
12
Notes:
Sr No
Biuret Assay
1
Reproducibility
2
Sensitivity
3
Accuracy
4
Range of Protein Concentration
5
Specificity
6
Interfering Agents
Low
Folin Lowry
Assay
Moderate
Bradford’s Assay
High
1 – 20 µg (Micro assay)
& 20 – 200 µg (Macro
Assay)
1 – 50 mg
Wide Range
Low
Moderate
High
Ammonium
Salts
Strong Acids ,
Buffers
Ammonium
Sulphate
Surfactants
PIHTBC005_O/0514
HTBC005-03
PROTEIN
13