EXPERIMENT 1: SPECTROPHOTOMETRIC PROTEIN ASSAYS

INTRODUCTION:
This experiment introduces the method of determining protein concentrations: absorbance at
540 nm, the Biuret, Lowry and Bradford assays. Determining the accurate protein
concentration is essential for all quantitative measurements of biochemical interactions.
Attributes of a good quantitation assay include the rapid, reliable and resistant to potentially
interfering substances. The students will see and evaluate these commonly-used assay
methods, and learn how to use spectrophotometer in measuring the absorbance of different
protein concentration.

METHODS:
A. Biuret Method
1. 1.5 mL of the Biuret reagent was added to each cuvette.
2. 1.0 mL of the unknown or protein standard was added to each cuvette.
3. The solution was mixed and incubated at room temperature for 30 minutes.
4. The OD was read at 555 nm.

B. Bradford Method
1. 3.0 mL of the Bradford reagent was added to each cuvette.
2. 0.06 mL of the unknown or protein standard was added to each cuvette.
3. The solution was mixed and incubated at room temperature for 5 minutes.
4. The OD was read at 595 nm.

C. Lowry Method
1. 0.25 mL of protein was mixed with 2.5 mL of Lowry reagent 1.
2. After 10 minutes, 0.25 mL of Lowry reagent 2 was added and the solution was
mixed well immediately.
3. After 30 minutes, the absorbance at 750 nm was measured.
RESULTS:

A. Biuret Method

BSA sample
Optical Density (OD)
(mg/mL)
Blank 0.000
2.0 0.533
4.0 0.602
6.0 0.623
8.0 0.625

Graph of Optical Density versus BSA sample

0.64
f(x) = 0.01 x + 0.52
0.62 R² = 0.79

0.6

0.58

0.56
OD

0.54

0.52

0.5

0.48
1 2 3 4 5 6 7 8 9
BSA sample (mg/mL)
B. Bradford Method

BSA sample
Optical Density (OD)
(mg/mL)
Blank 0.000
0.1 0.411
0.2 0.535
0.3 0.611
0.4 0.651

Graph of Optical Density versus BSA sample

0.7
f(x) = 0.8 x + 0.35
0.6 R² = 0.95

0.5

0.4
OD

0.3

0.2

0.1

0
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
BSA sample (mg/mL)
C. Lowry Method

BSA sample
Optical Density (OD)
(mg/mL)
Blank 0.000
0.1 0.049
0.2 0.042
0.3 0.066
0.4 0.180

Graph of Optical Density versus BSA sample

0.2
0.18
0.16
0.14 f(x) = 0.42 x − 0.02
0.12 R² = 0.69

0.1
OD

0.08
0.06
0.04
0.02
0
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
BSA sample (mg/mL)
DISCUSSION:
In this experiment, we had to determine the protein concentrations by Biuret, Lowry
and Bradford assays.
First, the Biuret method is the method that can be used to determine the amount of
soluble protein in a solution. It is very high and independent of composition amino acids.
This method least sensitive than Bradford method and Lowry method. The consistency of this
method is quite fair, and the linearity is linear. The Biuret reagent reacts with peptide bonds
and changes colour when it does so. The spectrophotometer has been used to measure the
intensity of the colour produced at the OD reading is taken at 555 nm. The more protein
present, the darker the colour. Based on the result, we can see that the OD reading 0.533 at
2.0 mg/mL which is at the lowest reading among all other. While, at 8.0 mg/mL, the OD
reading was 0.625 which is the highest reading. The graph shows that OD reading increases
as the concentration increment in linear standard curve. This proved that highest
concentration of protein has highest OD reading.
Then, the Bradford method is one such dye-based assay for protein concentration
estimation and it is the method that simpler, faster, more sensitive and very few interferences
by nonprotein components which is more convenience. This method is more sensitivity than
Lowry method and have good consistency. Bradford method is a non-linear concentration
dependence. The protein concentration can be evaluated by determining the amount of dye in
the blue ionic form and by measuring the absorbance at 595 nm. Based on the result, we can
see that the OD reading 0.411 at 0.2 mg/mL which is at the lowest reading among all other.
While, at 0.4 mg/mL, the OD reading was 0.651 which is the highest reading. The graph
shows that OD reading rise as the concentration adds in linear standard curve. This proved
that highest concentration of protein has highest OD reading.
Lastly, the Lowry method is one of the ways to determine the protein concentration in
a solution. This method is fairly convenience because it needs two-steps procedure that based
on two chemical reactions which requires minimum of 40 minutes incubation time, which
more time than other methods. The first reaction is the reduction of copper ions under
alkaline conditions, which is Biuret reaction. The second causes a colour change of the
solution into blue. The reading at 750 nm is recommended because at this wavelength, few
other substances absorb light which is directly proportional to the amount of protein in the
sample. This method is sensitive to low protein concentration and the consistency is
excellent. It is a non-linear concentration dependence. Based on the result, we can see that the
OD reading 0.042 at 0.4 mg/mL which is at the lowest reading among all other. While, at 0.4
mg/mL, the OD reading was 0.180 which is the highest reading. The graph shows that OD
reading not rise as the concentration increases in linear standard curve because 0.2 mg/mL
showed OD reading decreased which was 0.042. However, it proved that highest
concentration of protein has highest OD reading.
CONCLUSION:
In conclusions, we can determine the protein concentration by using these commonly-
used assay methods. All the assay methods showed that absorbance increment when the
concentration of the protein increased.

REFERENCES:
Anonymous. Protein Quantitation (2013). Retrieved on 23 September 2018 from
http://www.labome.com/method/Protein-Quantitation.html.
Kruger N.J. (1994). The Bradford Method for Protein Quantitation. In: walker J.M. (eds)
Basic Protein and Peptide Protocols. Methods in Molecular Biology TM, vol 32. Humana
Press. Retrieved on 23 September 2018
Olson B, Markwell J. Assays for determination of protein concentration. Curr Protoc Sci.
200;0: Unit 3.4. Retrieved on 23 September 2018.