Extraction and Isolation of Proteins
CALLO, Nisan May Date Due: Sept. 2, 2014 Date Submitted: Sept. 2, 2014
EXPERIMENT NO. 1
EXTRACTION AND ISOLATION OF PROTEINS
Background of the experiment
Pure proteins are not readily available for study or use. There are certain methods that should be done in order to obtain proteins from its sources. The objective of this experiment is to isolate casein from milk and albumin from egg and to identify and explain the principles behind the methods employed for protein extraction.
Results and Discussion
There are many possible sources where we could get proteins. It can be from plants, invertebrates and microorganisms. There are many purification strategies to obtain proteins but shown below is a general purification strategy for proteins.
Extraction --> Isolation --> Intermediate purification --> Final purification
In this experiment, egg and milk were the sources of proteins—albumin and casein. The extraction of albumin from egg white was done first. 20 mL of egg white and 2.0 mL of 1.0 M HOAc was mixed in order to make the solution to be around 4.55-4.88. This procedure is called isoelectric precipitation. Proteins have isoelectric points at which the charges of their amino acid side groups balance each other. If the ionic strength of a solution is either very high or very low proteins will tend to precipitate at their isoelectric point.[1] So the addition of acetic acid was done to in order to remove unwanted proteins through isoelectric precipitation. Before the real purification steps can begin, the protein must be released from the cells and subcellular organelles. Mechanical disruption was done by pressing the mixture against the sides of the funnel. This step, called homogenization, involves breaking open the cells.[2] The resulting mixture was then filtered through a damp cheesecloth to remove some impurities in the solution. The addition of (NH4)2SO4 was done to salt in albumin. This was slowly added to minimize saturating the
EXPERIMENT NO. 1
EXTRACTION AND ISOLATION OF PROTEINS
Background of the experiment
Pure proteins are not readily available for study or use. There are certain methods that should be done in order to obtain proteins from its sources. The objective of this experiment is to isolate casein from milk and albumin from egg and to identify and explain the principles behind the methods employed for protein extraction.
Results and Discussion
There are many possible sources where we could get proteins. It can be from plants, invertebrates and microorganisms. There are many purification strategies to obtain proteins but shown below is a general purification strategy for proteins.
Extraction --> Isolation --> Intermediate purification --> Final purification
In this experiment, egg and milk were the sources of proteins—albumin and casein. The extraction of albumin from egg white was done first. 20 mL of egg white and 2.0 mL of 1.0 M HOAc was mixed in order to make the solution to be around 4.55-4.88. This procedure is called isoelectric precipitation. Proteins have isoelectric points at which the charges of their amino acid side groups balance each other. If the ionic strength of a solution is either very high or very low proteins will tend to precipitate at their isoelectric point.[1] So the addition of acetic acid was done to in order to remove unwanted proteins through isoelectric precipitation. Before the real purification steps can begin, the protein must be released from the cells and subcellular organelles. Mechanical disruption was done by pressing the mixture against the sides of the funnel. This step, called homogenization, involves breaking open the cells.[2] The resulting mixture was then filtered through a damp cheesecloth to remove some impurities in the solution. The addition of (NH4)2SO4 was done to salt in albumin. This was slowly added to minimize saturating the
References: [1] Montelion, G. (2009). Protein purification. Proteomics and Functional Genomics. Retrieved from http://www-nmr.cabm.rutgers.edu/academics/biochem694/reading/ProteinPurification_Vermont.pdfApendix [2] Campbell, M. K. and Farrell, S. O. (2012). Biochemistry (7th Ed). Belmont, CA: Brooks/Cole. [3] Scopes, R.K. (1994). Protein purification: Principles and practice. New York, NY: Springer-Verlag New York, Inc. [4] Stenesh, J. (1984). Experimental biochemistry. New Jersey: Prentice Hall, Inc. [5] Spurlock, D. (2012). Isolation and identification of casein from milk. Retrieved from http://homepages.ius.edu/dspurloc/c122/casein.htm