EXPERIMENT NO.
-3: Qualitative estimation of protein using Biuret and Bradford
assay .
REQUIREMENTS:
HiPer Protein estimation Kit (Himedia) [contains Protein Standard, Biuret, Folin-Lowry,
Bradford Reagent], Test Tubes, Test Tubes Stand, Spectrophotometer, Cuvette, dH2O.
THEORY:
Principle:
1) Biuret Assay:
The biuret test, commonly known as “Piotrowski's test”. Biuret protein assay is based on
the principle that copper ions (Cu2+) react with peptide bonds in proteins, forming a
complex that absorbs light at a specific wavelength. The biuret reagent used in the assay
contains copper ions that react with the peptide bonds in the protein to form a purple-
colored complex. In this assay, the copper (II) binds with nitrogen atoms present in the
peptides of proteins.
Figure1:Schematic representation of Biuret assay
2. Bradford Assay:
Bradford Reagent contains a dye, Coomassie brilliant Blue G-250, which has an absorbance
maximum of 465 nm in unbound state. The Bradford protein assay is based upon the
formation of complexes between Coomassie Brilliant Blue G-250 and the protein samples
in solution.
When the protein sample binds to the dye the color of the solution turns blue from brown
and there is a shift in the absorption maximum of the dye from 465 nm to 595 nm. This dye
binding procedure is completed within 5 minutes and the blue colored complex formed is
stable for one hour. Thus, concentration of unknown protein sample can be derived by
plotting its absorbance value on the standard curve. The standard curve is obtained from
the absorbance readings of the series of standard protein dilutions assayed alongside the
unknown sample. (20-2000 ug/mL).
Figure2:Schematic representation of Bradford assay.
PROCEDURE:
● Eight sterilized test tubes were taken and labelled them as Blank, and 200 µL
Test (T1/T2).
● 200 µL of Protein sample-1 was taken in fresh test tube.
● Add the colorimetric reagent in both the blank and test samples and mix well.
● Follow the instructions for each assay as mentioned in table below.
● Reading was taken in spectrophotometer at 540 nm, 750 nm, and 595 nm.
● OD of all the test tubes were recorded after adjusting with blank.
Colorimetric Assay
Tube Amt. of
Amt. of reagent Incubation time Blank Test
Sample
Biuret (540 nm) 2 mL 200 µL 10 min at RT 0.00 0.320
Braford 200 µL 0.00 0.662
1 mL 10 min at RT
(595 nm)
OBSERVATIONS AND RESULTS:
Tube Blank Test
Biuret 0.00 0.320
Braford 0.00 0.662
PRECAUTION:
● Mix the reaction mixture properly before readings.
● Wipe the cuvette before taking readings.
EXPERIMENT NO.-4. Quantitative estimation of protein using Biuret and Bradford Assay.
REQUIREMENTS:
HiPer Protein estimation Kit (Himedia) [contains Protein Standard, Biuret, Bradford
Reagent], Test Tubes, Test Tubes Stand, Spectrophotometer, Cuvette, dH2O.
THEORY:
Principle:
1) Biuret Assay:
The biuret test, commonly known as “Piotrowski's test”. Biuret protein assay is based on
the principle that copper ions (Cu2+) react with peptide bonds in proteins, forming a
complex that absorbs light at a specific wavelength. The biuret reagent used in the assay
contains copper ions that react with the peptide bonds in the protein to form a purple-
colored complex. In this assay, the copper (II) binds with nitrogen atoms present in the
peptides of proteins.
Figure1: Biuret assay and condensation reaction of urea
PROCEDURE:
● Eight sterilized test tubes were taken and labelled them as Blank, 1, 2, 4, 6, 8, 10
mg/200 µL and Test (T1/T2).
● Dilutions of protein (BSA) were prepared with concentrations of 0.2, 0.4, 0.8, 1.2, 1.6,
2.0 mg/200 µL from stock (50 mg/mL). [1-4 µL, 2-8 µL, 4- 16 µL, 6-24 µL, 8-32 µL, 10-
40 µL]
● Volume was maintained to 200 µL with dH2O.
● 200 µL of Protein sample-1 was taken in fresh test tube.
● 2 ml of Biuret reagent was added in all test tubes including Protein sample-1/2 and
was mixed well.
● Then sample was kept at room temperature for 10 minutes.
● Reading was taken in spectrophotometer at 540 nm.
● OD of all the test tubes were recorded after adjusting with blank.
Tube No. Blank 1 2 3 4 5 6 7
Test
Conc. Of BSA (mg) 0 0.2 0.4 0.8 1.2 1.6 2.0
Sample-1/2
Amt of Stock (µL) 0 4 8 16 24 32 40 200 µL
Amt of dH2O (µL) 200 196 192 184 176 168 160 0
Amt of Biuret
2 2 2 2 2 2 2 2
reagent (ml)
Absorbance (OD) at 0.00 0.062 0.065 0.079 0.092 0.136 0.181 0.320
540 nm
DETERMINATION OF PROTEIN
CONCENTRATION OF UNKNOWN SAMPLE:
𝐶𝑜𝑛𝑐𝑛𝑒𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑢𝑛𝑘𝑛𝑜𝑤𝑛 𝑖𝑛 𝑚𝑔
𝑃𝑟𝑜𝑡𝑒𝑖𝑛 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑡𝑒𝑠𝑡 × 10=
𝑠𝑎𝑚𝑝𝑙𝑒 = 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛 µ𝐿
= (4.2/200)*10
𝑷𝒓𝒐𝒕𝒆𝒊𝒏 𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒊𝒏 𝒕𝒆𝒔𝒕 𝒔𝒂𝒎𝒑𝒍𝒆 = … …0.21 … … … . 𝒎𝒈/µL
2. Bradford Assay:
Bradford Reagent contains a dye, Coomassie brilliant Blue G-250, which has an absorbance
maximum of 465 nm in unbound state. The Bradford protein assay is based upon the
formation of complexes between Coomassie Brilliant Blue G-250 and the protein samples
in solution.
When the protein sample binds to the dye the color of the solution turns blue from brown
and there is a shift in the absorption maximum of the dye from 465 nm to 595 nm. This dye
binding procedure is completed within 5 minutes and the blue colored complex formed is
stable for one hour. Thus, concentration of unknown protein sample can be derived by
plotting its absorbance value on the standard curve. The standard curve is obtained from
the absorbance readings of the series of standard protein dilutions assayed alongside the
unknown sample. (20-2000 µg/mL).
Figure2: Schematic representation of Bradford assay.
PROCEDURE:
● Eight sterilized test tubes were taken and labelled them as Blank, 1 to 7.
● Dilutions of protein (BSA) were prepared with concentrations of 4, 8, 12, 16, 20
µg/200
µL from stock (1 mg/mL). [1-4 µL, 2-8 µL, 4- 16 µL, 6-24 µL, 8-32 µL, 10-40 µL]
● Volume was maintained to 200 µL with dH2O.
● 200 µL of Protein sample-1 was taken in fresh test tube.
● 1 ml of Bradford’s reagent was added in all test tubes including Protein sample-1/2
and was mixed well.
● Then sample was kept at room temperature (RT) for 10 minutes.
● Reading was taken in spectrophotometer at 595 nm.
● OD of all the test tubes were recorded after adjusting with blank.
Tube No. Blank 1 2 3 4 5 6
Test
Conc. Of BSA (µg) 0 4 8 12 16 20
Sample-1/2
Amt of Stock (µL) 0 4 8 12 16 20 200 µL
Amt of dH2O (µL) 200 196 192 188 184 180 0
Amt of Biuret
1 1 1 1 1 1 1
reagent (ml)
Absorbance (OD) at 0.00 0.151 0.278 0.287 0.330 0.662 0.224
540 nm
DETERMINATION OF PROTEIN CONCENTRATION OF UNKNOWN SAMPLE:
𝐶𝑜𝑛𝑐𝑛𝑒𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑢𝑛𝑘𝑛𝑜𝑤𝑛 𝑖𝑛 𝑚𝑔
𝑃𝑟𝑜𝑡𝑒𝑖𝑛 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑖𝑛 𝑡𝑒𝑠𝑡 × 5=
𝑠𝑎𝑚𝑝𝑙𝑒 = 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛 µ𝐿
= (10.1/200)*5
𝑷𝒓𝒐𝒕𝒆𝒊𝒏 𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒊𝒏 𝒕𝒆𝒔𝒕 𝒔𝒂𝒎𝒑𝒍𝒆 = … … 0.2525… … … . 𝒎𝒈/µL
RESULT:
The change in color confirms the presence of protein in the given sample. Protein
concentration was …………. mg/mL from biuret assay and ……… mg/mL by Bradford assay.
OBSERVATION:
PRECAUTION:
● Wipe the cuvette before taking readings.
