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UV Spectrophotometric Protein Assay

Section I: Introduction
a) Objective- The purpose of the experiment was to identify the protein concentration of
an unknown using Beer’s Law and spectrophotometers.

b) Theory- Spectrophotometers is based on the concept that ultraviolet lights can be used
to measure the absorbance value of proteins at specific wave lengths and in this case,
recorded at 280nm and 330nm. A calibrated curve can be produced with the data to
extrapolate an unknown’s concentration and in addition, Beer’s law can then be used to
calculate the unknown protein’s concentration.

Section II: Experimental

a) Reagents and Materials- 17.4 mg of bovine serum albumin (BSA) and 1.725g NaH2PO4
and DI water were used to generate specific concentrations of protein solutions.
Cuvettes were used to hold the solutions. An unknown protein concentration also used.
Volumetric pipets for 200uL and 1000uL were used to measure the specific quantities of
buffer to add.

b) Equipment: Shimadzu spectrometer was used to measure the BSA absorbance at wave
lengths 280nm and 330nm.

c) Procedure:
1) Calculate the needed amount of NaH2PO4 to create a phosphate buffer of 7.5pH with a
volume of 500mL and concentration of 25mM which is 1.725g of NaH2PO4.
a) The phosphate buffer has 1.725g of NaH2PO4 added and filled with DI water to
490mL. It was slowly titrated to a pH of 7.5 with a strong base and then filled to the
500mL marker with DI water.
2) Obtain a vial of BSA and add 1.5 mL of 25mM phosphate buffer to the vial and dissolve
protein completely to create a stock solution of BSA.
3) Calculate the needed amount of stock solution and phosphate buffers to create desired
concentrations of 75, 125, 225, & 500 ug/mL. Afterwards, create the desired
concentrations and add NaH2PO4 to the 5mL marker.
a)75ug/mL requires 32.33 uL of BSA solution
b)125ug/mL requires 53.88 uL of BSA solution
c) 225ug/mL requires 96.96 uL of BSA solution
d) 500ug/mL requires 215.52 uL of BSA solution
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4) Prepare the spectrophotometer by blanking it with a cuvette filled with 2mL of

phosphate buffer where absorbance values are measured at wavelengths 280nm and
330nm.
5) Rinse out the cuvette with DI water and wipe sides with wipes.
6) Add the concentration of 75ug/mL protein solution into the cuvette and run it through
the spectrophotometer
7) Repeat steps 5 and 6 again but instead of using 75ug/mL, use the other concentrations
and also the unknown.
8) Measure the unknown 3 times using three different aliquots.

Section III: Data and Calculations

a) Raw Data:

BSA Solution Concentrations Absorbance at

(ug/mL) 280nm Absorbance at 330nm
75 0.033644 0.0109495
125 0.0689186 0.00216782
225 0.182303 0.0151841
500 0.428855 -0.000576258
Unknown 1 0.0027776 -0.00523013
Unknown 2 0.004161 -0.00505203
Unknown 3 0.0135648 0.00311059

b) Calculation and Statistical Analysis:

Preparing phosphate buffer:
(138 g/mol) (25 mmol/L) (1mol/1000mmol) 0.5 L = 1.725 g

BSA Stock Solution:

17.4 mg / 1.5 mL = 11.6 mg/mL

Desired BSA Solutions:

V1 = (C2V2) / C1

V1 = ((.075mg/mL) (5mL))/11.6 mg/mL x1000 uL = 32.33 uL of BSA stock

V1 = ((.125mg/mL) (5mL))/11.6 mg/mL x1000 uL = 53.88 uL of BSA stock

V1 = ((.225mg/mL) (5mL))/11.6 mg/mL x1000 uL = 96.98 uL of BSA stock

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V1 = ((.500mg/mL) (5mL))/11.6 mg/mL x1000 uL = 215.52 uL of BSA stock

Corrected A280 values:

A280 (corrected) = A280 – (A330) (1.929)

75 µg/mL: A280 (corrected) = 0.033644– (.0109495) (1.929) = 0.012522415

125 µg/mL: A280 (corrected) = .0689186– (.00216782) (1.929) = 0.064736875

225 µg/mL: A280 (corrected) = .182303– (.0151841) (1.929) = 0.153012871

500 µg/mL: A280 (corrected) = .428855– (-.00576258) (1.929) = 0.439971017

Unknown 1: A280 (corrected) = 0.00277776– (-0.00523013) (1.929) = 0.012866681

Unknown 2: A280 (corrected) = 0.004161– (-0.00505203) (1.929) = 0.013906366
Unknown 3: A280 (corrected) = 0.013564– (0.00311059x 1.929) = 0.007563672

Unknown Concentrations
Concentration = (absorbance + .035)/ .0009
Unknown 1: (0.012866681+.035)/ .0009=53.19ug/mL
Unknown 2: (0.013906366+ .035)/ .0009=54.34ug/mL
Unknown 3: (0.007563672+ .035)/ .0009=47.29 ug/mL
Mean: (53.19+ 54.34+47.29)/3 = 51.61ug/mL
Standard Deviation:
([(51.61-53.19) ^2+(51.61-54.34) ^2+(51.61-+47.29) ^2]/ (3-1))^.5= 3.78
Report Scientific Results: 51.61 ± 3.78
% Relative standard deviation: (3.78/51.61)100%= 7.32%
Q Test: 47.29, 53.19, 54.34
Qcalc: abs (53.19-47.29)/( 54.34-47.29) = .84
Qtab of 90% confidence= .94
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Since Qtab > Qcalc, we do not reject 47.29.

95% confidence interval:
df=3-1=2
51.61± 4.30(3.78)/(3)^.5= 42.23 & 60.99
=51.61±9.38
Beer’s Law Calculation of unknown
Absorbance average: (0.012866681+0.013906366+0.007563672)/3= .01
Concentration=Absorbance/[(extinction coefficient)(path length)]
C= .01 ÷[43284M^-1cm^-1(1cm)]= 2.31 x 10^-7M
2.31 10^-7mol/L x 66400g/mol x 1L/1000mL x 10^6ug/g= 15.3384ug/mL=.0153384mg/mL

c)Final Data
Corrected Absorbance at 280nm Graph and Data

Corrected
BSA Solution Concentrations Absorbance at
(ug/mL) 280nm
0 0
75 0.012522415
125 0.064736875
225 0.153012871
500 0.428855
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Corrected Absorbance at 280nm

0.5

0.45

0.4
Corrected Absorbance at 280nm

f(x) = 0.000901812017844113 x − 0.0350097911011609

R² = 0.982467677635542
0.35

0.3
Corrected Absorbance at 280nm
0.25 Linear (Corrected Absorbance at
280nm)
0.2

0.15

0.1

0.05

0
0 100 200 300 400 500 600

BSA Solution Concentrations (ug/mL)

Linear Regression Analysis obtained with Microsoft Excel software:

Slope= .0009
Y-intercept = -.035
Absorbance = .0009(Concentration) - .035
Concentration = (absorbance + .035)/ .0009
R² = 0.9825

Section IV: Results and Discussion

a) Significance of Data and Question 5: The unknown solution was found to have a
concentration of 51.61 ± 3.78ug/mL according to the linear regression (calibration
curve) developed from the Shimadzu spectrophotometer’s data. In contrast, according
to Beer’s Law, it was derived that the concentration was 15.3384ug/mL. There was a
large difference between the two methods and calls into question the quality of the
spectrophotometer or Beer’s Law. However, it’s worth noting the %rsd was 7.32% so
the data points were quite precise. It is also known that there is 95% confidence interval
of 42.23ug/mL and 60.99ug/mL. The sources of error may be that the cuvettes were not
properly wiped or there were small miscalculations and roundings in terms of math.
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b) Objective Achieved: The objective of finding was successful since the concentrations of
the unknown was found using Beer’s Law and a linear regression model.

c) References
Boyer , Rodney F. “Modern Experimental Biochemistry: 3rd Edition” Prentice Hall
(August 2, 2001).