2013-48660 September 3, 2014

September 3, 2014

EXPERIMENT NO. 1

EXTRACTION AND ISOLATION OF PROTEINS

Background of the experiment

The experiment aimed to acquire and isolate casein from both whole and non-fat milk, and albumin from egg. The methods followed were based on various principles of protein extraction from which a simple purification technique may be applied.

Results and Discussion

The extraction process of casein from 20mL non-fat milk started by obtaining the pH of the sample, which was 6, adding 0.1M HCl afterwards until the occurrence of a flocculent precipitate, and then checking the pH again, which resulted to 5. Checking the initial pH is essential because calcium caseinate (salt form of casein present in milk) normally has a pH of 4.6, meaning it is insoluble in solutions with pH less the mentioned. The milk, having a pH of 6, gives casein a negative charge and is solubilized as a salt. The addition of HCl aims to neutralize the casein micelles through protonation causing these micelles to degenerate and for the protein casein to precipitate from the solution, observed as flocculent precipitate. It also aids in reaching the isoelectric point (pH 4.6) of the protein to remove the charges and inhibit them from repelling from each other that will promote coagulation, which is a characteristic of proteins. These techniques relate to the structure of proteins being pH dependent and amphoteric, wherein they form salts with both cation and anion based on the net charge. Afterwards, the obtained solution was transferred into two pre-weighed centrifuge tubes, each having equal amount of the solution and uniformly distributed precipitate. These underwent centrifugation to properly separate and isolate the precipitate (protein casein) from the supernatant (the rest of the components of the non-fat milk sample). This technique depicts the homogeneity of proteins. The extracted and isolated casein was then washed twice with 95% ethanol to get rid of any remaining unwanted fat from the residue and since casein (polar) is insoluble in ethanol, the product yield will not be altered. Afterwards, the precipitate was washed with acetone to remove excess glutaraldehyde which immobilizes enzymes that would alter the normal biological mechanism of the protein. The residue then, was air dried under the hood to eliminate excess water present. The residue from both pre-weighed centrifuge tubes were weighed and were found to be 7.80g and 7.72g, respectively. Lastly, the obtained casein was dissolved in 0.1M NaOH for storage. The calculated percentage (w/v) yields were 11.2% and 10.4%, these values give a close approximate on how much protein (casein) is present per volume of non-fat milk. These residues were labeled and stored in the refrigerator for future experiments.

Table 1. Data on Extraction of Casein from Non-Fat Milk

Measurement Centrifuge Tube A Centrifuge Tube BInitial pH of milk 6pH after addition of 0.1M HCl 5Weight of casein (residue) 1.12g 1.04gTotal volume of casein solution 10mLPercentage (w/v) yield of casein solution 11.2% 10.4%

The theoretical protein content of the non-fat milk sample was 13% which was relatively higher than our experimental yields which were 11.2% and 10.4%. There might be minimal errors while transferring the solution

(solution with flocculent precipitate) into the two centrifuge tubes, there might still be precipitate existing in the beaker which was thought to be bubbles from moderate stirring of the milk sample, which might have decreased the percentage yield. There might also be a small amount of residue decanted together with the supernatant or rinsing reagents. Being vigilant and cautious throughout the experiment and during measurements may improve the yield and purity of the extracted casein.

There are several techniques on protein extraction and since these proteins are commonly contained in protein bodies present inside the cell, cell disruption or lysis by either chemical or physical means is necessary to acquire them. Extraction methods vary in procedures among microorganisms, plants and animals because these organisms differ in cellular components and cellular permeability characteristics. And as mentioned a while ago, extraction methods depend on protein properties, which has a wide variety per creature, and that sets another distinction among the stated organisms.

Summary, Conclusions, and Recommendations

Casein and albumin extraction underwent through different extraction procedures because these two are different types of proteins—globular and fibrous, respectively, which vary in shape and structure, thus they also differ in extraction methods. The type of organism, their cellular characteristics, and physical properties of the proteins they contain play an important role in determining the procedure of extraction to be performed in order to successfully acquire ample protein from the sample (in our case, casein from non-fat milk).

References

Seyhan, E. (1996). Chem2O06 - 1997/98 - Experiment 11. Retrieved August 28, 2014, from http://www.chemistry.mcmaster.ca/~chem2o6/labmanual/expt11/2o6exp11.html

Migneault, I., Dartiguenave, C., Bertrand, M. J., & Waldron, K. C. (2004). Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. BioTechniques, 37(5), 790–802.

Shimizu, M., & Yamauchi, K. (1981). Isolation and Characterization of Mucin-Like Glycoprotein in Human Milk Fat Globule Membrane. The Journal of Biochemistry,91(2), 515-524.

Schilling, E. A., Kamholz, A. E., & Yager, P. (2002). Cell Lysis and Protein Extraction in a Microfluidic Device with Detection by a Fluorogenic Enzyme Assay. Analytical Chemistry, 74(8), 1799-1804. doi:10.1021/ac015640e

Appendix

Weight of centrifuge tubes = 6.68g (each pre-weight centrifuge tube)

Weight of casein residue (with centrifuge tube)

A = 7.80g B = 7.72g

Weight of casein (residue only)

A = 7.80g – 6.68g B = 7.72g – 6.68g

A = 1.12g B = 1.04g

Casein solution A %(w/v) Casein solution B %(w/v)

= 1.12g / 10mL = 1.04g / 10mL

= 11.2% = 10.4%