UNIVERSITI TEKNOLOGI MARA FAKULTI KEJURUTERAAN KIMIA CHEMICAL ENGINEERING LABORATORY (CHE465) No. Title Allocated Marks (%) Marks 1 Abstract/Summary 5 2 Introduction 5 3 Aims 5 4 Theory 5 5 Apparatus 5 6 Methodology/Procedure 10 7 Results 10 8 Calculations 10 9 Discussion 20 10 Conclusion 5 11 Recommendations 5 12 Reference / Appendix 5 13 Supervisor’s grading 10 TOTAL MARKS 100 Remarks: 1 NAME : MARISSA DE VALDA BT MOHD YATIM STUDENT NO : 2013229382 GROUP : GROUP 1 EXPERIMENT : ESTIMATION OF PROTEIN DATE PERFORMED : 15 OCTOBER 2014
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UNIVERSITI TEKNOLOGI MARA FAKULTI KEJURUTERAAN KIMIA
1. All series were included a zero protein (water) tube (reagent blank).
2. Lowry: 0.25 mL of protein was mixed with 2.5 mL of Lowry’s reagent. After 10
minutes, 0.25 mL of Lowry reagent 2 was added and mixed well immediately.
After 30 minutes, the absorbance was measured at 750 nm.
3. Bradford: 0.25 mL of protein was mixed with 2.5 mL of Bradford’s reagent and
the absorbance was measured at 595 nm.
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b) Lowry Reagents
Reagent 1:
One volume of reagent B (0.5% copper sulfate pentahydrate, 1% sodium or potassium tartrate) was mixed with 50 volumes of reagent A (2% sodium carbonate, 0.4% NaOH).
Reagent 2:
Commercial Folin-Ciocalteu phenol reagent was diluted with an equal volume of water.
To quantify protein: 0.25 mL of protein was mixed with 2.5 mL of Lowry’s reagent. After 10 minutes, 0.25 mL of Lowry reagent 2 was added and mixed well immediately. After 30 minutes, the absorbance was measured at 750 nm.
c) Bradford's Reagent
100 mg Coomassie Blue G-250 was dissolved in 50 mL of 95% ethanol, and 100 mL of 85% phosphoric acid was added before being diluted to one liter. The reagent was filtered once as it seems to precipitate dye over time.
To quantify protein: 0.25 mL of protein was mixed with 2.5 mL of Bradford’s
reagent and the absorbance was measured at 595 nm after 5 minutes.
Disadvantages: A high blank which may affect subsequent readings because some reagent adheres to the cuvette. Another is that it is very sensitive to the presence of detergent e.g. from poorly-rinsed glassware.
d) Data Analysis
Separate graphs for each assays were plotted based on the data obtained.
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5. RESULTS
A. Lowry’s Reagent Method
i. Bovine Serum Albumin ( BSA )
Concentration(mg/ml)
Absorbance
0.00 0.000
0.12 0.401
0.16 0.504
0.20 0.507
0.24 0.676
0.28 0.585
0.30 0.511
10
0 0.05 0.1 0.15 0.2 0.25 0.3 0.350
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
R² = 0.761884638363958
Absorbance vs Concentration of BSA
Concentration of BSA (mg/ml
Abso
rban
ce
ii. Gelatin
Concentration(mg/ml)
Absorbance
0.0 0.000
0.2 0.097
0.4 0.227
0.6 0.235
0.8 0.368
1.0 0.506
11
0 0.2 0.4 0.6 0.8 1 1.20
0.1
0.2
0.3
0.4
0.5
0.6
R² = 0.970021580124689
Absorbance vs Concentration of Gelatin
Concentration of Gelatin (mg/ml)
Abso
rban
ce
The strong blue colour is created by two reactions that is first, the formation of the
coordination bond between peptide bond nitrogens and a copper ion and secondly, the reduction
of the Folin-Ciocalteu reagent by tyrosine (phosphomolybdic and phosphotungstic acid of the
reagent react with phenol). The measurement is carried out at 750 nm. A calibration curve is
created and the concentration of the unknown protein is determined from the curve [2].
B. Bradford’s Reagent Method
i. Bovine Serum Albumin ( BSA )
Concentration(mg/ml)
Absorbance
0.00 0
0.12 0.412
0.16 0.649
0.20 0.773
0.24 0.994
0.28 1.120
12
0.30 1.136
0 0.05 0.1 0.15 0.2 0.25 0.3 0.350
0.2
0.4
0.6
0.8
1
1.2R² = 0.991522377713978
Absorbance vs Concentration of BSA
Concentration of BSA (mg/ml
Axis
Title
ii. Gelatin
Concentration(mg/ml)
Absorbance
0.0 0.000
0.2 0.289
0.4 0.547
0.6 0.879
0.8 0.936
1.0 1.120
13
0 0.2 0.4 0.6 0.8 1 1.20
0.2
0.4
0.6
0.8
1
1.2R² = 0.966907449269794
Absorbance vs Concentration of Gelatin
Concentration of Gelatin (mg/ml
Axis
Title
Bradford assay, a colorimetric protein assay, is based on an absorbance shift of the dye
Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is
converted into its bluer form to bind to the protein being assayed. During the formation of this
complex, two types of bond interaction take place: the red form of Coomassie dye first donates
its free electron to the ionizable groups on the protein, which causes a disruption of the protein's
native state, consequently exposing its hydrophobic pockets. These pockets in the protein's
tertiary structure bind non-covalently to the non-polar region of the dye via van der Waals forces,
positioning the positive amine groups in proximity with the negative charge of the dye. The bond
is further strengthened by the ionic interaction between the two. The binding of the protein
stabilizes the blue form of the Coomassie dye; thus the amount of the complex present in
solution is a measure for the protein concentration, and can be estimated by use of an absorbance
reading [5].
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C. Spectophotometry Assay
i. Bovine Serum Albumin ( BSA )
Concentration(mg/ml)
Absorbance
0.00 0.000
0.12 0.019
0.16 0.142
0.20 0.177
0.24 0.242
0.28 0.327
15
0.30 0.359
0 0.05 0.1 0.15 0.2 0.25 0.3 0.350
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
R² = 0.901632561377876
Absorbance vs Concentration of BSA
Concentration of BSA (mg/ml)
Abso
rban
ce
ii. Gelatin
Concentration(mg/ml)
Absorbance
0.0 0.000
0.2 0.004
0.4 0.035
0.6 0.044
0.8 0.078
1.0 0.081
16
0 0.2 0.4 0.6 0.8 1 1.20
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
R² = 0.953340456288103
Absorbance vs Concentration of Gelatin
Concentration of Gelatin (mg/ml)
Abso
rban
ce
This method is based on the fact that two of the aromatic amino acids, tryptophan and
tyrosine, show a peak in absorbance around 280 nm. It has the advantage of being quick and
easy. Since it needs no chemical reaction to be performed, it is widely used for detection of
proteins or peptides during their separation by chromatography. As proteins contain different
ratios of aromatic amino acids, per se it is more suited to the comparison of solutions of the same
protein and less to absolute measurement. The latter requires the knowledge of the molar
extinction coefficients of proteins. For many proteins, these were determined and can be found in
the literature. Moreover, if we know the number of tyrosine and tryptophan amino acids in the
protein of interest, since their absorption values are additive, it is possible to calculate the molar
extinction coefficient [2].
6. CALCULATION
a. Bovine Serum Albumin (BSA)
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Concentration
(mg/ml)
Volume of BSA
(ml)
Volume of Water
(ml)
0.00 0 250
0.12 30 220
0.16 40 210
0.20 50 200
0.24 60 190
0.28 70 180
0.32 80 170
b. Gelatin
Concentration
(mg/ml)
Volume of Gelatin
(ml)
Volume of Water
(ml)
0.00 0 100
0.20 20 80
0.40 40 60
0.60 60 40
0.80 80 20
1.00 100 0
7. CONCLUSION AND RECOMMENDATION
From the findings of this experiment, it can be concluded that most commercial protein
assay reagents are well-characterized, robust products that provide consistent, reliable results.
Nevertheless, each assay reagent has its limitations; having a basic understanding of the
chemistries involved with each type of assay is essential for selecting an appropriate method for
a given sample and for correctly evaluating results.
Bradford assay
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Bradford assays are the fastest and easiest to perform of all protein assays. The assay is
performed at room temperature and no special equipment is required. Resultant blue color is
measured at 595nm following a short room temperature incubation. The Coomassie dye-
containing protein assays are compatible with most salts, solvents, buffers, thiols, reducing
substances and metal chelating agents encountered in protein samples.
The main disadvantage of Coomassie based protein assays is their incompatibility with
surfactants at concentrations routinely used to solubilize membrane proteins. In general, the
presence of a surfactant in the sample, even at low concentrations, causes precipitation of the
reagent. In addition, the Coomassie dye reagent is highly acidic, so proteins with poor acid-
solubility cannot be assayed with this reagent. Finally, Coomassie reagents result in about twice
as much protein-to-protein variation as copper chelation-based assay reagents.
The recommendation is that the ready-to-use liquid Coomassie dye reagents should be
mixed gently by inversion just before use. The dye in these liquid reagents forms loose
aggregates within 60 minutes in undisturbed solutions. Gentle mixing of the reagent by inversion
of the bottle will uniformly disperse the dye and ensure that aliquots are homogeneous.
Lowry Assay
The assay is performed in two distinct steps. First, protein is reacted with alkaline cupric
sulfate in the presence of tartrate for 10 minutes at room temperature. During this incubation, a
tetradentate copper complex forms from four peptide bonds and one atom of copper (this is the
"biuret reaction"). Second, a phosphomolybdic-phosphotungstic acid solution is added. This
compound (called Folin-phenol reagent) becomes reduced, producing an intense blue color.
The final blue color is optimally measured at 750nm, but it can be measured at any
wavelength between 650nm and 750nm with little loss of color intensity. It is best to measure the
color at 750nm since few other substances absorb light at that wavelength.
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The recommendation is that the Folin phenol reagent must be added to each tube
precisely at the end of the ten minute incubation. At the alkaline pH of the Lowry reagent, the
Folin phenol reagent is almost immediately inactivated. Therefore, it is best to add the Folin
phenol reagent at the precise time while simultaneously mixing each tube. The Modified Lowry
Protein Assay Reagent must be refrigerated for long-term storage, but it must be warmed to room
temperature before use. Using cold Modified Lowry Protein Assay Reagent will result in low
absorbance values.
Spectrophotometric Assay
This method is based on the fact that two of the aromatic amino acids, tryptophan and
tyrosine, show a peak in absorbance around 280 nm. It has the advantage of being quick and
easy. Since it needs no chemical reaction to be performed, it is widely used for detection of
proteins or peptides during their separation by chromatography. As proteins contain different
ratios of aromatic amino acids, per se it is more suited to the comparison of solutions of the same
protein and less to absolute measurement. The latter requires the knowledge of the molar
extinction coefficients of proteins. For many proteins, these were determined and can be found in
the literature. Moreover, if we know the number of tyrosine and tryptophan amino acids in the
protein of interest, since their absorption values are additive, it is possible to calculate the molar
extinction coefficient.
REFERENCES
[1] Petrucci, Ralph H. General Chemistry: Principles and Modern Applications. Macmillian:
2007.
[2] Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein
utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976.
[3] Berg, J.M., Tymoczko, J.L., Stryer, L.: Biochemistry (2012) 7th edition, W. H. Freeman and
Company, New York; ISBN-13: 9781429229364
20
[4] Krohn, R.I. (2002). The Colorimetric Detection and Quantitation of Total Protein, Current
Protocols in Cell Biology , A.3H.1-A.3H.28, John Wiley & Sons, Inc.
[5] Dennison, Clive (2003), A guide to protein isolation, Focus on structural biology 3: 39, ISBN