Protein Concentration Determination using Spectrophotometer

This laboratory report explains the experiment conducted to determine the concentration of different samples of proteins using a spectrophotometer. The report covers the aim, introduction, method, results, calculations, discussion, and conclusion of the experiment. The apparatus used in the experiment includes standard bovine serum albumin (BSA) solution, cuvettes, 1.5 ml centrifuge tubes, spectrophotometer, Bradford reagent, pipettes, and water. The report also provides references for further reading.

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Title: Protein Concentration
Abstract
This experiment aimed at determining the concentration of different samples of proteins.
Different methods are used in the determination of the concentration of protein in solution
samples. In this experiment, a spectrophotometer was used and a calibration curve generated
from the values obtained. The sample solutions illustrated different color changes. The darker the
color the higher the protein concentration in the sample. A line of best fit was generated and the
equation in the form y=mx+c was generated in which y represented the absorbance of the
samples at 595nm.
Aim
To determine the concentration of different samples of proteins using ultraviolet spectroscopy
Introduction
The determination of the concentration of proteins in a sample is a fundamental step when it
comes to any laboratory workflow that revolves around extraction and/ or analysis of proteins.
The knowledge on the amount of proteins contained in a sample is useful in making comparison
of results from one protein yield to another (Buxbaum, 2015, p.178). The knowledge is as well
important in the accurate measurement of the kinetics of enzymes and normalization of various
samples for the purposes of comparison or storage. The choice of an appropriate assay has a
significant role to play when it comes to the determination of the concentration of protein in a
sample (Cote, 2010, p.275).

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Estimation of the concentration of a given protein sample is one of the most commonly tasks
carried out in biochemistry lab. Numerous methods are deployed in the determination of the
concentration of proteins including analysis of the changes in the properties of the spectra in
some dyes in the presence of proteins, analysis of amino acids following hydrolysis of the acid
protein as well as spectrophotometric estimation of the proteins either in far or near UV region
(Kurtz, 2009, p.215).
The main objective in this lab is to come up with the curves for each of the BSA standard
absorbances against the concentration in mg/mL in order to determine the concentration of the
proteins for the unknown samples. Multiple assays can be conducted to determine the
concentration of a protein sample but not all the samples would be appropriate for the specific
data that is required for this lab (Michael, 2011, p.335). Biuret Assay is ideal for proteins that
have low concentration ranging from 5 to 16 mg/mL and uses copper (II) in binding the peptide
bonds thereby turning the solution to violet. The absorption in Biuret Assay is measured at
540nm.
Apparatus
 Standard bovine serum albumin (BSA) solution (1 mg/mL)
 Cuvettes
 1.5 ml centrifuge tubes
 Spectrophotometer (Richard, 2011, p.198)
 Bradford reagent
 Pipettes
 Water

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 Unknown proteins samples A, B and C
Method
Part 1: Making a protein concentration standard curve
1. Turn on the spectrophotometer and set the wavelength to 595 nm (Whitney, 2010, p.188)
2. Prepare a series of dilutions of your BSA standard solution of concentration between 0.1
mg/mL and 0.8 mg/mL (Roger, 2010, p.415)
3. To a 1.5 mL centrifuge tube, add 0.5 mL of the protein sample and 1.5 mL of Bradford
reagent. Mix by inversion and let the tube stand for 5 minutes
4. After 5 min, pour the contents of the centrifuge tube into a cuvette and measure the
absorbance of the samples in the spectrophotometer at 595 nm (using 0 mg/mL protein
sample as your blank)
Part 2: Determine the protein concentration of unknown samples A, B and C
1. Add Bradford reagent to the unknown samples as you did in part 1 (Satinder, 2010,
p.239)
2. Measure the samples at 595 nm and record their absorbance
Part 3: Analysis of results
1. Create a standard curve using the values you obtained from part 1. Once a standard curve
has been made, you can use this work out of the protein concentration of your unknowns.
Results
Concentration BSA Water Abs

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1 100 0 0.298
0.80 80 20 0.317
0.60 60 40 0.212
0.40 40 60 0.162
0.20 20 80 0.033
0.10 10 90 0.011
0.0 0 100 0
A 0.237
B 0.413
C 0.236
Graph
Calculations

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The expression that is generated by the linear trend of the best fit is used in the determination of
the concentration of the unknown samples A, B and C. in the line of best fit trend, the y-intercept
represents the absorbance while the x-intercept represents the concentration (Seung, 2016,
p.162). The units that are in correspondence with the slope are in mg/mL and the equation for the
slope is y=0.336× 0.0079 ^a
Discussion
The main objective of this experiment was to determine the protein concentration of unknown
samples A, B and C. it was evident that there was a color change upon the addition of Bradford
reagent to the samples (Michael, 2011, p.255). While there was instantaneous color change in
some of the samples where the color changed instantly to dark blue, some of the samples
remained light blue in color. A darker color was an indication of higher concentration of the
proteins in the sample.
A calibration curve is produced through plotting of the concentrations of the unknown samples
against the readings of their absorbance values. From the plotted values, a line of best fit is
generated that gives an equation in the form y=mx+c in which y represents the absorbance at
595nm and x the represents the concentration of the proteins going by the data obtained from the
averages of the two absorbance sets measured (Donglu, 2012, p.218). One of the samples
generated negative readings of the absorbance indicating that the concentration of the proteins is
that sample was way below that of the control sample that was used in the experiment.
From the obtained results of the experiment, it can be concluded that the concentrations of the
unknown samples A, B and C were 0.237 mg/mL, 0.413 mg/mL and 0.236 mg/mL respectively.
The absorbance data was then analyzed using a 95% confidence interval and generated an error

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margin of +/- 0.001% with the R-values from the experiment being approximately 1 thereby
increasing the accuracy levels of the equation (Burden, 2012, p.186). The R-values also show
that the estimated values on the concentration of proteins will be reflective values that are close
to the actual concentrations of the proteins whose concentrations are unknown and are to be
determined (Sushanta, 2016, p.189).
Conclusion
In conclusion, spectrophotometer is one of the methods that can be used in the estimation of the
concentration of proteins in a solution. In conjunction with other reagents such as Bradford
reagent, the experiment provided grounds on measurement of protein concentration on quite a
reliable scale. In this experiment, the concentrations of three unknown samples of proteins were
tested to determine their protein concentration. From the obtained results and the plotted graph,
sample B had the high concentration of proteins while sample C contained the least
concentration

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References
Burden, D.W., 2012. Biotechnology Proteins to PCR: A Course in Strategies and Lab
Techniques. 2nd ed. New York: Springer Science & Business Media.
Buxbaum, E., 2015. Fundamentals of Protein Structure and Function. 2nd ed. Manchester:
Springer.
Cote, E., 2010. Clinical Veterinary Advisor - E-Book: Dogs and Cats. 2nd ed. Manchester:
Elsevier Health Sciences.
Donglu Zhang, S.S., 2012. ADME-Enabling Technologies in Drug Design and Development.
2nd ed. Beijing: John Wiley & Sons.
Kurtz, S.M., 2009. HMWPE Biomaterials Handbook: Ultra High Molecular Weight
Polyethylene in Total Joint Replacement and Medical Devices. 3rd ed. Kansas: Academic Press.
Michael D. Willard, H.T., 2011. Small Animal Clinical Diagnosis by Laboratory Methods - E-
Book. 5th ed. OXford: Elsevier Health Sciences.

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Richard A. McPherson, M.R.P., 2011. Henry's Clinical Diagnosis and Management by
Laboratory Methods E-Book. 22nd ed. New York: Elsevier Health Sciences.
Roger L. Lundblad, F.M., 2010. Handbook of Biochemistry and Molecular Biology, Fourth
Edition. 4th ed. London: CRC Press.
Satinder Ahuja, S.S., 2010. Handbook of Modern Pharmaceutical Analysis. 4th ed. Kansas:
Academic Press.
Seung-Beom Hong, M.B.R.L.Z.S.-V., 2016. Methods in Biotechnology. 2nd ed. Sydney: John
Wiley & Sons.
Sushanta K. Mitra, S.C., 2016. Microfluidics and Nanofluidics Handbook: Fabrication,
Implementation, and Applications. 5th ed. London: CRC Press.
Whitney, D.B., 2010. Med Lab Tech Vol 1, 2/e. 3rd ed. New York: Tata McGraw-Hill Education.