Experiment 1: SPECTROPHOTOMETRIC PROTEIN ARRAY

INTRODUCTION

One commonly used method for determining the total protein in a sample is the Biuret
method. The Biuret method is based on the complexation of Cu2+ to functional groups in the
protein’s peptide bonds. The formation of a Cu2+-protein complex requires two peptide
bonds and produces a violet-coloured chelate product which is measured by absorption
spectroscopy at 540 nm.

The Bradford method is based on the proportional binding of the dye Coomassie to proteins.
Within the linear range of the protein, the more protein present, the more Coomassie binds.
Furthermore, the assay is colorimetric; as the protein concentration increases, the color of the
test sample becomes darker. Coomassie absorbs at 595 nm.

Lowry’s reaction in which the Folin Ciocaltaeu reagent, which contains phosphomolybdic
complex which is a mixture of sodium tungstate, sodium molybdate and phosphate, along
with copper sulphate solution and the protein forms a blue purple colour which can be
assessed by measuring the absorbance at 650-700nm.

METHOD & PROCEDURES

Analytical Procedure

a. A540:Measure the absorbance of 1 mL protein at 540 nm

1.5 mL of the protein standard added to each tube and the OD at 540 were read.

b. Bluret Method

-Procedure [Protein range 0.125 mg/mL to 2.50 mg/mL]-

1. 1.5 mL of the Biuret reagent added to each tube.

2. 1.0 mL of protein standard added to each tube.

3. The sample were mix and incubated at room temperature for 30 minutes.

4. The OD at 555 nm (1 cm path) was read.

c. Bradford Method

-Procedure [Protein range 0.100 mg/mL to 1.50 mg/mL]-

1. 3.0mL of the Bradford reagent added to each tube.

2. 0.06 mL of protein standard added to each tube.
 3. Mixed and incubated at room temperature for 5 minutes.
4. The OD at 595 nm (1 cm path) were read.

d. Lowry Method

1. 0.25 mL of protein mixed with 2.5 mL of Lowry reagent I

2. After 10 minutes, 0.25 mL of Lowry reagent 2 added and mixed well immediately.
3. After 30 minutes, the absorbance measured at 750 nm.

RESULT

b. Biuret Method (0.096 blank)

[BSA] mg/mL OD
2.0 0.562
4.0 0.539
6.0 0.636
8.0 0.606

OD vs BSA Concentration at 555 nm

0.66
0.64
0.62
0.6 f(x) = 0.11 x + 0.53
R² = 0.46
0.58
OD
OD

0.56
Linear (OD)
0.54
0.52
0.5
0.48
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
[BSA]
c. Bradford Method

[BSA] mg/mL OD
0.1 0.442
0.2 0.447
0.3 0.507
0.4 0.474

OD vs BSA Concentration at 595 nm

0.52

0.5

0.48 f(x) = 0.16 x + 0.43

R² = 0.46
0.46 OD
OD

Linear (OD)
0.44

0.42

0.4
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
[BSA]

d. Lowry Method (0.055 blank)

[BSA] mg/mL OD
0.1 0.171
0.2 0.217
0.3 0.468
0.4 0.570

OD vs BSA Concentration at 600 nm

0.6
f(x) = 1.45 x − 0.01
0.5 R² = 0.94
0.4

0.3 OD
OD

Linear (OD)
0.2

0.1

0
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
[BSA]
DISCUSSION

The highest convenience among the three method is Bradford Method. It is because this
method is simple and faster than the other two. Single incubation only takes 5 minutes.
Bradford Method also compatible with wide range of substances and dependent on amino
acid. The second convenience is Biuret Method. Its independent of composition amino acid.
It is taking 30 minutes for single incubation and good general protein assay for batches of
material for which not having problem to yield. The least convenience is Lowry Method
because it involve two steps and taking long time. For double incubation take 40 minutes
total which are 10 minutes for first step and 30 minutes for second step.

Meanwhile, according to sensitivity, the most sensitive is the Bradford Method as it can
detect 1 to 100 mg/mL of protein concentration. The next one is Lowry Method as can detect
20 to 300 mg/mL of protein concentration. The least sensitive is Biuret method as 1 to 10
mg/mL of protein concentration detection.

Based on the generality ( the consistent of the result), Lowry Method shows the best
consistency while the Biuret Method for the second and least consistency for the Bradford
Method.

Last but not least, based on the linearity of the graph, only Biuret Method is linear
concentration dependent while the other two, Bradford Method and Lowry Method are non-
linear concentration dependence.

CONCLUSION

In conclusion, from this experiment, we are able to know the three methods of protein
concentrations. We are able to determine which method is the most simplest and efficient
method, which one is the most sensitive or maybe based on their generality or linearity.

REFERENCES

 Waterborg, J. H. (2002). The Lowry method for protein quantitation. In The protein
protocols handbook (pp. 7-9). Humana press.
 http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/c
h04s05.html
 https://www.analiticaweb.com.br/newsletter/16/51859_proteina_biureto.pdf