LAB 3 - SPECTROPHOTOMETRY

( PROTEIN ANALYSIS )

INTRODUCTION

Spectrophotometry is a procedure that frequently utilized in biological lab using

spectrophotometer which is a vital machine in studying various substance and understanding

the function and output results. Spectrophotometry is the measurement of the interaction of

light with substances. In this lab, we are using spectrophotometer to measure the absorbance

of a protein by a solution of light of specific wavelength ( 550 nm ). There are many ways to

measure protein concentration, one of the common way is by measuring the absorbance of a

coloured product formed by the protein and an organic molecule. In this practical lab, we are

using biuret test. Biuret test used for detecting the presence of peptide bond. When the peptide

bonds present, copper ion ( Cu2+ ) forms violet-colored in an alkaline solution. The protein

that mixed with biuret reagent then measured using spectrophotometry at 550 nm to determine

the absorbance.

OBJECTIVE

 To determine protein concentration using biuret protein assay with different sample.

 To determine the absorbance of protein with different concentration using

spectrophotometer
METHODOLOGY

1. First of all, the test tube are labelled as test tube 1, 2, 3, 4, 5, 6 and 7.

2. Each test tube filled with different volume of gelatine and water to get different

concentration of protein.

3. For test tube 1, it is containing only water and act as a blank or control.

4. For test tube 2, 0.1 mL of gelatin and 0.9 mL H2O was added.

5. For test tube 3, 0.2 mL of gelatin and 0.8 mL H2O was added.

6. For test tube 4, 0.3 mL of gelatin and 0.7 mL H2O was added.

7. For test tube 5, 0.4 mL of gelatin and 0.6 mL H2O was added.

8. For test tube 6, 0.5 mL of gelatin and 0.5 mL H2O was added.

9. For test tube 7, 0.6 mL of gelatin and 0.4 mL H2O was added

10. For our sample, 2 aditional tube were required. we used chicken eggs and quail eggs.

11. We take 1 mL of each sample and diluted with 19 mL of water to reduce its

concentration. Then, 1 mL of diluted sample was transferred to new test tube.

12. Then 2 mL of biuret reagent was added to each test tube.

13. The solutions were left for about 15 minutes before a small amount of each solution

transferred into cuvette.

14. Using spectrophotometry of specific wavelength ( 550 nm ), the absorbance reading

was taken.

15. Lastly, using the absorbance and protein concentration data, a standard curve can be

constructed.
 RESULT

Test tube Gelatin (mL) H2O (mL) Protein concentration Biuret Absorbance
1 0 1.0 0 0
2 0.1 0.9 1 0.079
3 0.2 0.8 2 0.144
4 0.3 0.7 3 2 mL 0.192
5 0.4 0.6 4 0.276
6 0.5 0.5 5 0.317
7 0.6 0.4 6 0.396
8 Sample X 0.254
9 Sample Y 0.423

Absorbance vs Protein concentration

0.5
0.45
0.4
0.35
absorbance

0.3
0.25
0.2
0.15
0.1
0.05
0
0 1 2 3 4 5 6 7
protein concentration
DISCUSSION

In this experiment, we want to determine the absorbance of the protein with

known concentration and determine the protein concentration of 2 different sample based

on its absorbance value. From the data, we had plottted a graph of absorbance vs protein

concentration. based on the graph, we can say that the absorbance is proportional to the

protein concentration. as the protein concentration increase, the absorbance value also

increased.

From the experiment that we perform, the absorbance value for chicken egg is

0.254 and from the graph the protein concentration is 3.9. While the absorbance value

for quail eggs is 0.463 and the protein concentration is 6.4. With this, we can say that the

quail egg contain higher protein than chicken egg. Quail eggs also has low calories but

are high in cholestrol. So we can suggest that we should eat quails eggs in moderation.
 7 test tube of solution and 2 test tube of sample

Absorbance value been taken using spectrophotometer

CONCLUSION

Throughout this lab, we can conclude that spectrophotometry and Biuret protein assay is

a valid and effective method to determine protein concentration and its absorbance value.

From this experiment, the protein concentration of unknown solutions were able to be

determined from its absorbance value. This experiment should be extended further to

analyse various others unknown solution.

REFERENCES

 https://www.scribd.com/doc/41425746/Protein-Lab-Report-Experiment-3

 www.augusta.edu/scimath/biology/.../biuretproteinassay.pdf

 http://biochemistrygirls.blogspot.my/2013/04/experiment-2-protein-

experiment.html