Extraction of Proteins

Topics: Bradford protein assay, Reducing sugar, Amino acid Pages: 8 (2007 words) Published: July 27, 2010
Biochemistry Laboratory (BCM 362L), Experiment No. 3, © February 14, 2006 3nd Quarter A.Y. 2005-2006

Extraction and Characterization of Proteins

Mr. *****1, *****2, *****2

1 Professor, School of CHE-Chm, Mapua Institute of Technology 2 Student, BCM 362L/ A31, School of CHE-Chm, Mapua Institute of Technology ____________________________________________________________



Invertase, Albumin and Casein were extracted from yeast, egg and milk respectively using solvent extraction, salting out and isoelectric precipitation. Benedict’ test was performed to determine the presence of reducing sugars in the prepared solution. Concentrations of albumin and casein were determined by two methods – Warburg-Christian method and Bradford Assay. By using the Warburg-Christian method, the concentration of casein is 0.3329 mg/mL. Different concentrations for different absorbance was computed using Beer’s law for the Bradford assay method, a linear plot was calibrated.

Keywords: extraction, assay, isoelectric, calibrated.



Proteins are the most diverse and abundant macromolecules within cells. Their functions range from the enzymes that carry out the numerous metabolic processes of the cell to structural components that gives cells structure and organization. One of the most useful parameters used in the study of proteins in solution is the concentration of the protein and. It is necessary for biochemists to estimate the concentration of proteins in a solution of interest in order to quantitatively determine the activity of the protein. In one part of the experiment, Benedict’s test was used. Here, monosaccharides and disaccharides can be detected because of their free aldehyde groups, thus, testing positive for the Benedict's test. Such sugars act as a reducing agent, and is called a reducing sugar. By mixing the sugar solution with the Benedict's solution and adding heat, an oxidation-reduction reaction will occur. The sugar will oxidize, gaining an oxygen atom, and the Benedict's reagent will reduce, loosing an oxygen atom. There are two types of spectrophotometric methods for determining protein concentration, which will be used for albumin and casein. The first method is called the Warburg-Christian method. In this method the protein concentration is estimated by making a direct absorption measurement of a solution in the UV range. The advantage of this method is that it is direct and it is nondestructive. The method also has disadvantages. First, the absorption at 280 nm will vary from protein to protein depending on the content of tyrosine and tryptophan.. Therefore, absolute quantitation of protein concentration by this method can only be determined for pure preparations of protein. Second, any substance that absorbs at 280 nm will interfere with the reading. However, the method can be quite useful as a rapid and nondestructive method to compare the relative concentrations of numerous protein solutions, such as the fractions generated during protein purification by column chromatography. The sensitivity of the assay varies with the tyrosine and tryptophan content of the protein solution, but is in the range of 1 mg protein/ml to 1 mg protein/ml. The second method to determine protein concentration is the protein dye-binding assay or Bradford assay. The dye, Coomasie brilliant blue, is a relatively hydrophobic substance that binds avidly to the hydrophobic regions of proteins. The change in absorption can be used to quantitate indirectly the amount of protein in the solution. The color change can be quantified with a spectrophotometer. There are two major advantages to the Bradford assay: 1) the ease of performance and 2) few interfering substances. The only interfering substances are high concentrations of detergents that disrupt the binding of the...

Cited: 1. Biochemistry, Garrett and Grisham, 3rd Edition
2. © 1993-2003 Microsoft Corporation. All rights reserved.
3. http://tecn.rutgers.edu/bio301s/Lab%203-%20protein%20diagram.htm
Continue Reading

Please join StudyMode to read the full document

You May Also Find These Documents Helpful

  • Extraction and Characterization of Proteins Essay
  • Extraction and Isolation of Proteins Essay
  • Essay on Protein Deficiency Symptoms
  • Extraction Essay
  • Proteins Essay
  • Protein Purification Essay
  • Protein Essay
  • Proteins Essay

Become a StudyMode Member

Sign Up - It's Free