Estimination of Protein 2
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Transcript of Estimination of Protein 2
TABLE OF CONTENT
No. Title pages
1 Abstract/Summary 2
2 Introduction 3
3 Aims 4
4 Theory 4
5 Methodology/Procedure 5
6 Appatatus 7
7 Results 9
8 Calculations 10
9 Discussion 16
10 Conclusion 17
11 Recommendations 17
12 Reference 18
13 Appendices 18
1
ABSTRACT
This experiment is conducted to learn the principles of protein assays. This exercise
introduces students to method of determining protein concentrations by using several method
which are lowry method, Bradford method and Biuret method.. This lab activity is designed
to teach students the principles behind a common protein estimation assay and identify the
efficiency and sensitivity of these three method of estimination of proteins. In this
experiment, there are two types of proteins have been used which are gelatine and bovine
serum albumin (BSA)
Standard Biuret reagent is prepared. The 0.50mL of protein is mixed with the
prepared Biuret Reagent. The solution is mixed well and allows standing for 10 minute. The
absorbance for each tube is read against the blank at 540 nm. The standard curve is plotted
using concentration of standard (mg/mL) against the absorbance at 540 nm.
Protein assays based on Lowry methods are divided into two categories, dye binding
protein assays and protein assays based on alkaline copper. Under alkaline conditions cupric
ions (Cu2+) chelate with the peptide bonds resulting in reduction of cupric ions (Cu2+) to
cuprous ions (Cu). The Cuprous ions can also be detected with Folin Ciocalteu Reagent
(phosphomolybdic/phosphotungstic acid) and this method is commonly referred to as the
Lowry method. Cuprous ions (Cu+) reduction of Folin Ciocalteu Reagent produces a blue
color that can be read at 750nm. The amount of color produced is proportional to the amount
of peptide bonds. In this experiment ,0.5 ml of protein is added to 5 ml of lowry reagent #1
and after 10 min,lowry reagent #2 is added and mix well. The mixture is leave for 30
minutes before using it
For Bradford method it is relies on the binding of Coomassie Brilliant Blue G250 dye
to protein, in which protein concentration is proportionate to the dye. In this experiment, 0.5
ml of protein with different concentration which are 0.02,0.04,0.06,0.08,0.10,0.12,0.14 and
0.16 are diluted with 5 ml of Bradford reagent The solution is mixed well and allows standing
for 10 minute. The absorbance for each tube is read against the blank at 595 nm. The standard
curve is plotted using concentration of standard (mg/mL) against the absorbance at 595 nm.
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Sensitivity is measured on how well does it detect small amounts of protein; for
instance, what mass of protein is needed to give an absorbance of 0.01The sensitivity increase
as the amount of protein detected increase. Based on this experiment, the most efficient and
sensitive is Bradford method because it has the highest amount of protein. The amount of
protein for gelatine is 41.82 mg while for BSA is 38.6 mg. The second efficient and sensitive
is Biuret method which the amount of protein for gelatine is 5.72 mg while for BSA is 14.1
mg. The lest sensitive and efficient method is Lowry method because contain the lowest
amount of protein. The amount of protein for gelatine is mg while for BSA is 38.6 mg. The
result for Biuret and Lowry is slightly different from the theory, this happen might because
of some error
INTRODUCTION
The protein in solution can be measured quantitatively by different methods. The methods
have been used in this experiment are Biuret method, Bradford method and Lowry method.
And in this experiment there are two types of protein have been used which are bovine serum
albumin (BSA) and gelatine.
The methods described by Bradford uses a different concept-the protein‘s capacity to bind to
a dye, quantitatively. The assay is based on the ability of proteins to bind to coomassie
brilliant blue and form a complex whose extinction coefficient is much greater than that of
free dye.
Protein assays based on Lowry methods are divided into two categories, dye binding protein
assays and protein assays based on alkaline copper. Under alkaline conditions cupric ions
(Cu2+) chelate with the peptide bonds resulting in reduction of cupric ions (Cu2+) to cuprous
ions (Cu). The Cuprous ions can also be detected with Folin Ciocalteu Reagent
(phosphomolybdic/phosphotungstic acid) and this method is commonly referred to as the
Lowry method.
For biuret method,In alkaline solutions, cupric ion complexes with the peptide bonds of
proteins and peptides to form a purple charge transfer complex (λmax = 540 nm). The
intensity of the color is proportional to the protein concentration. This reaction occurs only
with the peptide bond and not with the amino acid side chains. Because the number of
peptide bonds per given unit weight is approximately the same for all proteins, this method is
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generally applicable and reasonably accurate, irrespective of the composition of the protein
mixture
AIMS
This experiment is conducted:
1. To learn the principles of protein assays.
2. To determine protein concentrations by using several method which are lowry
method, Bradford method and biuret method..
3. To identify the efficiency and sensitivity of these three method of estimination of
proteins
THEORY
Biuret method determination of protein determination
In alkaline condition ,copper (II) is thought to bind to the peptide nitrogens of proteins and
this complex absorb light maximally at 550nm.in addition ,it is thought that in biuret reaction
some of the Cu²+ is reduce to Cu + in a secondary reaction with proteins
Figure 1 putative cupric complex with peptide bond of proteins
Lowry method of protein determination
The lowry method is commonly used method of protein determination because it is
inexpensive, easy to perform , sensitive, and highly reproducible Lowry is based on copper
complex formation with the peptide bond. Lowry methods are divided into two categories,
dye binding protein assays and protein assays based on alkaline copper. Under alkaline
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conditions cupric ions (Cu2+) chelate with the peptide bonds resulting in reduction of cupric
ions (Cu2+) to cuprous ions (Cu). The Cuprous ions can also be detected with Folin Ciocalteu
Reagent (phosphomolybdic/phosphotungstic acid) and this method is commonly referred to
as the Lowry method. Cuprous ions (Cu+) reduction of Folin Ciocalteu Reagent produces a
blue color that can be read at 750nm. The amount of color produced is proportional to the
amount of peptide bonds
Bradford method of protein estimination
The methods described by Bradford uses a different concept-the protein‘s capacity to bind to
a dye, quantitatively the one of the most commonly used method of protein determination
because it is easy to perform , very sensitive. The assay is based on the ability of proteins to
bind to coomassie brilliant blue and form a complex whose extinction coefficient is much
greater than that of free dye. Bradford is influenced by pH of your protein solution (not only
due to the protonation state of your charge residue side chain but also the dye has different
absorbance maxima at different pH).
METHODOLOGY
Preparation of stock solution
1. The gelatin or bsa which in form of powder is dilute with distilled water to form
stock solution of 0.16 mgml .
2. the mass of powder need to be added is calculated by ,
stock solution =mass of powder
Volume of water
Preparation of Lowry reagent
Lowry reagent #1
One volume of 500 ml (2.0 % sodium carbonate and 0.4% sodium hydroxide) is
diluted with 50 volume of 10 ml (0.5 % copper sulphate and 1.0 %sodium tartarate)
5
Lowry reagent # 2
5 ml of folin abcatteu phenol is diluted with 5ml of distilled water
Preparation of blank sample
1. Lowry assay: o.5 ml of distilled water is diluted with 5 ml of lowry reagent 1 .then,
after 10 minutes, 0.5 ml of lowry reagent 2 is added and mixed well.leave it for 30
minutes before using it.
2. Biuret assay : one ml of distilled water is diluted with five ml of biuret reagent
3. Bradford assay : 0.5 ml of distilled water is diluted with five ml of Bradford reagent.
Biuret method
1. The stock solution either BSA sample or gelatin is diluted with distilled water for the
concentration 0.02,0.04,0.06,0.08,0.10,0.12,0.14,0.16.
2. The amount of distilled water need to be added to the stock solution was calculated
by using this formula
m1 v1=m2 v2
3. 1.0 ml of sample is diluted with 5.0 ml of biuret reagent
4. The solution is mixed well and allows standing for 10 minute
5. The absorbance for each tube is read against the blank at 540 nm
6. The standard curve is plotted using concentration of standard (mg/mL) against the
absorbance at 540 nm
Bradford method
1. The stock solution either BSA sample or gelatin is diluted with distilled water for the
concentration 0.02,0.04,0.06,0.08,0.10,0.12,0.14,0.16.
2. The amount of distilled water need to be added to the stock solution was calculated
by using this formula
m1 v1=m2v2
3. 0.5 ml of sample is diluted with 5.0 ml of Bradford reagent
4. The solution is mixed well and allows standing for 10 minute
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5. The absorbance for each tube is read against the blank at 595 nm
6. The standard curve is plotted using concentration of standard (mg/mL) against the
absorbance at 595 nm
Lowry method
1. The stock solution either BSA sample or gelatin is diluted with distilled water for the
concentration 0.02,0.04,0.06,0.08,0.10,0.12,0.14,0.16.
2. The amount of distilled water need to be added to the stock solution was calculated
by using this formula
m1 v1=m2v2
3. 0.5 ml of sample is diluted with 5.0 ml of Lowry reagent #1
4. The solution is mixed well and allows standing for 10 minute
5. After 10 minutes0.5 ml of Lowry reagent #2 is added
6. The solution is leave for 30 minutes
7. The absorbance for each tube is read against the blank at 7500 nm
8. The standard curve is plotted using concentration of standard (mg/mL) against the
absorbance at 750 nm
APPARATUS
Proteins: bovine serum albumin (BSA) and gelatine
Distilled water
Biuret Reagent
1. 10% (w/v) NaOH
2. 0.3% copper sulfate pentahydrate
3. 1.2% sodium potassium tartrate
4. potassium iodide
Lowry Reagents
1. Reagent 1:
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Reagent B (0.5% copper sulfate pentahydrate, 1% sodium or potassium tartrate)
Reagent A (2% sodium carbonate, 0.4% NaOH)
2. Reagent 2:
Folin-Ciocalteu phenol reagent
Bradford's Reagent
1. Coomassie Blue G-250
2. 95% ethanol
3. 85% phosphoric acid
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RESULT
gelatin sample
Concentration of
gelatine mg/ml
Method
Biuret assay Bradfort assay Lowry assay
0.02 0.032 0.480 0.852
0.04 0.042 0.607 0.705
0.06 0.034 0.507 0.886
0.08 0.039 0.705 1.114
0.10 0.033 0.713 2.037
0.12 0.046 0.740 0.920
0.14 0.032 0.623 0.681
0.16 0.036 0.621 3.300
BSA sample
Concentration of
gelatine
Method
Biuret assay Bradfort assay Lowry assay
0.02 0.034 1.518 1.090
0.04 0.043 1.610 1.674
0.06 0.044 1.655 2.104
0.08 0.046 1.689 2.508
0.10 0.050 1.704 2.695
0.12 0.051 1.723 3.002
0.16 0.054 1.762 3.252
0.16 0.052 1.763 3.073
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CALCULATION
Biuret method in gelatine sample
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.180
0.0050.01
0.0150.02
0.0250.03
0.0350.04
0.0450.05
f(x) = 0.161904761904762 x + 0.0209166666666667
Absorbance at 540 nm vs Concentration of mixture
Y-ValuesLinear (Y-Values)
concentration of sample (mg/ml)
Absobance at 540 nm
Y= mx + c, where y=0.01
Y=0.1619x +0.0209
X=0.01-0.0209
0.1619
=0.0673 mg/ml
Amount of protein
=0.0673 mg/ml x 85ml
=5.72mg
10
Bradford method in gelatine sample
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.180
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
f(x) = 1.05595238095238 x + 0.529464285714286
Absorbance at 595 nm vs Concentration of mixture
Y-ValuesLinear (Y-Values)
concentration of sample (mg/ml)
Absobance at 595 nm
Y= mx + c, where y=0.01
Y=1.056x +0.5295
X=0.01-0.5295
1.056
=0.492 mg/ml
Amount of protein
=0.492 mg/ml x 85ml
=41.82 mg
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Lowry method in gelatine sample
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.180
0.5
1
1.5
2
2.5
3
3.5
f(x) = 9.13095238095238 x + 0.395464285714286
Absorbance at 750 nm vs Concentration of mixture
Y-ValuesLinear (Y-Values)
concentration of sample (mg/ml)
Absobance at 750 nm
Y= mx + c, where y=0.01
Y=9.131x +0.3955
X=0.01-0.3955
9.131
=0.0422 mg/ml
Amount of protein
=0.0422 mg/ml x 85ml
=3.95 mg
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Biuret method in bsa sample
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.180
0.01
0.02
0.03
0.04
0.05
0.06f(x) = 0.146428571428571 x + 0.0342857142857143
Absorbance at 540 nm vs Concentration of mixture
Y-ValuesLinear (Y-Values)
concentration of sample (mg/ml)
Absobance at 540 nm
Y= mx + c, where y=0.01
Y=0.1464 x +0.0343
X=0.01-0.0343
0.1464
=0.1659 mg/ml
Amount of protein
=0.1659 mg/ml x 85ml
=14.1 mg
13
Bradford method in bsa sample
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.180
0.20.40.60.8
11.21.41.61.8
2
f(x) = 3.10357142857143 x + 1.35367857142857
Absorbance at 595 nm vs Concentration of mixture
Y-ValuesLinear (Y-Values)
concentration of sample (mg/ml)
Absobance at 595 nm
Y= mx + c, where y=0.01
Y=3.1036x + 1.3537
X=0.01-1.3537
3.1036
=0.433 mg/ml
Amount of protein
=0.433 mg/ml x 85ml
=36.8 mg
14
Lowry method in bsa sample
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.180
0.5
1
1.5
2
2.5
3
3.5f(x) = 17.3803571428571 x + 0.941714285714286
Absorbance at 750 nm vs Concentration of mixture
Y-ValuesLinear (Y-Values)
concentration of sample (mg/ml)
Absobance at 750 nm
Y= mx + c, where y=0.01
Y=17.38x +0.9417
X=0.01-0.9417
17.38
=0.0536 mg/ml
Amount of protein
=0.0536 mg/ml x 85ml
=4.557 mg
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DISCUSSION
This experiment is done to apply the general approach which is to determine the
amount of protein by using several method which biuret method ,Bradford method and lowry
method, and to identify the efficiency and sensitivity by using three method of estimination
of protein. There are two types of proteins have been used which are gelatine and bovine
serum albumin (BSA)
In this experiment, the graph of absorbance at 540nm versus concentration of
sample(mg/ml) for Biuret, the graph of absorbance at 595 nm versus concentration of
sample (mg/ml) for Bradford and graph of absorbance at 750 nm versus concentration of
sample (mg/ml) for Lowry are plotted for both protein. Based on this experiment for the
generality ,the result among two different proteins are consistent because the result for BSA
sample and gelatine quite similar for Bradford method and Lowry method, but for Biuret the
result among the protein is not consistence because the result quite different which for
gelatine sample is 5.72 mg while for BSA sample is 14.1 mg. For the linearity ,all the graph
gives a straight line, but for the gelatine sample even though the graph gives the straight line
but some of the point is alternating for Biuret method while for Lowry method and Bradford
method the point quite not consistent.
For sensitivity based on the theoretical states that the most sensitive method is
Bradford method followed by the Lowry method and the less sensitive is Biuret method.
Sensitivity is measured on how well does it detect small amounts of protein; for instance,
what mass of protein is needed to give an absorbance of 0.01The sensitivity increase as the
amount of protein detected increase. Based on this experiment, the most efficient and
sensitive is Bradford method because it has the highest amount of protein. The amount of
protein for gelatine is 41.82 mg while for BSA is 38.6 mg. The second efficient and sensitive
is Biuret method which the amount of protein for gelatine is 5.72 mg while for BSA is 14.1
mg. The lest sensitive and efficient method is Lowry method because contain the lowest
amount of protein. The amount of protein for gelatine is mg while for BSA is 38.6 mg. The
result for Biuret and Lowry is slightly different from the theory, this happen might because
of some error .
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There are several source of error need to be consider .first, parallax error occur while
carrying this experiment because the eye is not perpendicular to the readings of measuring
cylinder. Second, the reagents expose to the air because some of the reagents are volatile.
Next, using the unclean and contaminate apparatus that make the result less accurate
especially for cuvettes because the spectrophotometer is really sensitive. Besides, using the
unspecific tissue while wiping the cuvette, this can make some particle stick to the cuvette
since the spectrophotometer is sensitive.
CONCLUSION
As conclusion, this experiment is carryout successfully. The objective which to determine the
amount of protein by using several method which Biuret method ,Bradford method and
Lowry method ,and to identify the efficiency and sensitivity by using three method of
estimination of protein are achieved. Based on this experiment, Bradford method is the most
sensitive and efficient method since it can detect large amount of protein at absorbance 0.01
for BSA and gelatine sample. The amount of protein detected for gelatine is 41.82 mg while
for BSA is 36.8 mg. Unfortunately for Biuret method and Lowry method is slightly different
from the theoretical this might be due to some error while carrying this experiment.
RECOMMENDATION
Use the clean apparatus to ensure the result of the experiment is not disturb by
unknown solution and to prevent from the contamination
Avoid parallax error and make sure the eyes is perpendicular to the readings of the
measuring cylinder
Ensure that to handle carefully cuvette bottle for accuracy and prevent contamination.
We have to handle cuvette with only gloves and touch only the areas not in the light
path.
Ensure that using specific tissue while wiping the cuvettes to get the accurate result
Besides that, the mixture of sample of either gelatin or bsa are dilute with water
properly and both are shaken for several times to make sure the homogenous mixture
formed.
ensure that there is no air bubbles trapped in pipette because it can make the result
less accurate.
REFERENCES
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1. Gornall, A.G., Bardawill, C. J. and David, M. M. (1949)Determination of
Serum Proteins by means of the Biuret Reaction.J. Biol. Chem. 177; 751.
2. Layne, Ennis Spectrophotometric and Turbidimetric Methods for Measuring
Proteins.Methods in Enzymology, vol. 3, p. 447 (1957)
3. Peterson, Gary L.Determination of Total Protein.Methods in Enzymology,
vol. 91, p. 95 (1983)
APPENDICES
Figure 2:gelatin sample with different concentration
Figure 3:Bradford method
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