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Isolation and Qualitative Tests of Proteins
I. Objectives
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 prepared) • Bradford Reagents; BSA standard (100uh/mL)
• 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
B. Qualitative Tests
C. Protein Assay Using the Bradford Method
3. Bradford Assay
Computed Concentration:
VI. Discussion
VII. Conclusion
- 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.
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
September 6, 2018
Date Submitted: