Laboratory Report No.

3
Isolation and Qualitative Tests of Proteins
 
 
Esconde, Yvonne Keithlene J.  BS Nursing 1A
Grp. 3 / Thurs (7:00–7:30 PM)      Aug. 30, 2018   
Rating: ________
 
 
I. Objectives
 
1. To isolate the protein, casein, in skimmed milk through isoelectric precipitation.
2. To test for the specific chemical groupings on the protein structure of a casein from skimmed
milk.
 
II. Introduction
 
Proteins are large organic compounds made of amino acids arranged in a linear chain
and joined together by peptide bonds between the carboxyl groups and amino groups
of adjacent amino acid residues.
In order to perform a vitro analysis, a protein must be purified away from other
molecular components. A mixture can be purified using ultracentrifugation,
precipitation (salting out), chromatography, electrophoresis, spectroscopy, or by
enzyme assays.
A number of qualitative color reactions have been devised which are useful for
detection of proteins. These tests are used with the knowledge that they test for the
specific chemical groupings on the protein structure.
III. Materials
 
A. Equipment
• (2) 250-mL Beaker
• (2) 5-mL Pipette
• (5) 10-mL Test Tubes
• Litmus Paper
• Filter Paper
• Hot Plate
• pH meter or pH Indicator
 
B. Reagents
• Skimmed Milk • 0.01M CuSo4
• Hopkins-Cole Reagent • 2.5M NaOH
• Millon’s Reagent (freshly • Bradford Reagents; BSA standard (100uh/mL)
prepared)
• 0.1% Ninhydryin Solution • 0.1M HCl
• 10% NaOH • 0.02% Naphtol Solution
 • Conc. H2SO4 • 2% NaOBr (freshly prepared)
• Conc. HNO3 • Evaporated Milk Sample
• Conc. NaOH • UV-Vis Spectrophometer
 
 
IIII. Methodology (Schematic Diagram)
A. Isoelectric Precipitation of Casein
 
 
 
 
 
 
 
 
 
 
 
 
 
 
B. Qualitative Tests
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
C. Protein Assay Using the Bradford Method
 
Test Tube 1 2 3 4 5 6 7 8 9
No.
mL Standard 0 0.1 0.1 0.20 0.2 0.3 0.35 0.4 0.45
0 5 5 0 0
mL H2O 1.5 1.4 1.3 1.30 1.2 1.2 1.15 1.1 1.05
0 5 5 0 0
 
V. Data and Results
1. Isolation of Protein
Weight of Casein:
Physical Appearance:

2. Qualitative Tests for Proteins

 
Qualitative Tests Observations
1. Biuret Test Clear greenish liquid with a purple ring on top
2. Ninhydrin Test No changes in color
3. Xanthoproteic Test Yellow and yellow orange color reactions
4. Millon’s Test Coagulated and formed pink color reaction
5. Hopkins-Cole Test 2 layers: dark brown below, white foam above
6. Sakaguchi Test 3 layers: yellow-brown on top, clear middle, milky white below
7. Nitroprusside Test Bright yellow
8. Fohl’s Test Yellowish and then yellowish brown

3. Bradford Assay 
 
Absorbance Concentration of Protein
10x Dilution
50x Dilution
100x
Dilution
 
Linear Regression Equation:
 
 
 
 
 
Computed Concentration:
 
 
 
 
 
 

VI. Discussion
 
 
 
 
 
 
 

VII. Conclusion
 
 
 
 
 
 
 
 
 
VIII. Assessment (Q&A)
1. What is meant by the isoelectric point of a protein?
- This is where the protein achieves neutrality, or when it has no net charge.
 
2. At what pH is a protein least soluble? Why?
 
- A protein has its lowest solubility at its isoelectric point, which is usually within the range of
5.5 to 8. The net charge (either positive or negative) of a protein can interact with water
molecules and disperse the protein molecules more easily. At isoelectric point, the protein has no
net charge, and thus, is least soluble on it.
 
3. What type of chemical grouping is present in all proteins?
- Amino acids
- NH2
- COOH
 
4. Give the principle involved and the chemical structure responsible for the positive Biuret test,
Ninhydrin test, Xanthoproteic test, Millon’s test, Hopkins-Cole test, and Sakaguchi test.
 
-
 
 
  
 
 
 
 
 
IX. References
 
Dr. Sundin. (n.d.) Protein and amino acid tests. Proteins and amino acids. Retrieved
fromhttps://people.uwplatt.edu/~sundin/351/351h-pro.htm
 Wang, N. S. (n.d.). Enzyme purification by isoelectric precipitation. Biochemical
engineering laboratory. Retrieved from https://eng.umd.edu/~nsw/ench485/lab6c.htm
X. Certification / Conforme
 
 

Esconde, Yvonne Keithlene J. 

Signature over printed name
 
September 6, 2018
Date Submitted: