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Extraction and Isolation of Proteins

University/College: University of Arkansas System
Date: October 28, 2017
Words: 985
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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 Hoax was mixed in order to make the solution to be around 4. 55-4. 88. This procedure is called sclerotic precipitation. Proteins have sclerotic 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 sclerotic point. [l] So the addition of acetic acid was done to In order to remove unwanted proteins through Sclerotic precipitation. Before the real purification steps can begin, the protein must be released from the cells and subculture organelles. Mechanical disruption was done by pressing the mixture against the sides of the funnel. This step, called homogeneities, 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 (NH)SASS was done to salt in albumin. This was slowly added to minimize saturating the solution that can lead to salting out albumin. Though filter systems are commonly used for separating precipitates, it is not enough to thoroughly separate them so a centrifuge is used. [3] Centrifugation refers to the process of subjecting either a solution to a centrifugal force in order to separate the components of a solution or suspension. [4] After centrifugation, the precipitate was discarded and the supernatant was Immersed in an ice bath to prevent the denunciation of proteins.

NH)SASS was then added to the supernatant until the solution appears turbid. A turbid solution Indicates that albumin has been salted out. Salting out is largely dependent on the hydrophobic of the protein, interactions with the solvent. The salt (NH)SASS was used in the salting in and salting out process because it is highly water-soluble, its ions does not disrupt protein bonding, it is not affected with temperature, it has high ionic strength, and it provides some crude purification of proteins away from non-proteins and also separates some protein. (NH)SASS also yields a precipitated protein slurry that is usually very stable. L] In the extraction of casein from milk, non-fat milk was used to prevent proteins from precipitating with the casein. The higher the fat in milk, the lower the % purity will be and vice versa. Isolation of casein is achieved by acidification. 0. 1 M HCI was added to 15 ml milk until it curdles. This was done to decrease the pH to about 4. Calcium casein has an sclerotic point of pH 4. 6. This means it is insoluble in solutions with a pH less than 4. 6. The pH of milk is 6. 6, therefore, casein has a negative charge at this pH and is soliloquies as a salt.

If an acid is added to milk, the negative charges on the outer surface of the casein micelles are neutralized, by preparation of the phosphate groups. The casein micelles are destabilize or aggregate because the electric charge is decreased to that of the sclerotic point. The casein micelles disintegrate and the casein (the neutral protein) precipitates because it is no longer polar, with the calcium ions remaining in solution. [5] ca+castanets + CLC came + acacia (1) Casein has an metaphoric nature, thus excess acid is avoided because it would dissolve casein which would result to a lower yield of extracted casein.

After the acidification, the solution was placed in the centrifuge to separate casein from other unwanted components. The supernatant was discarded and the precipitate was washed with 95% ethanol. This is done to precipitate and remove lactose from the precipitate. The precipitate was also washed with acetone to remove lipids present in it. The crude casein and albumin extract was weighed. Their calculated % (w/v)’s were 16. 73% for casein and 12. 33% for albumin. Protein extraction procedures vary according to its source because a different type of cell has a different purification method.

There are differences in protein extraction procedures among microorganisms, plants and animals. Animal tissues vary from very easily broken erythrocytes to tough collagen’s material such as found in blood vessels and other smooth-muscle containing tissue. Plant cells are generally more difficult to disrupt than animal cells because of the celluloid cell walls. Bacteria vary from fairly fragile organisms that can be broken up by digestive enzymes or osmotic shock. [3] Summary, Conclusions and Recommendations Based on the results, the albumin and casein was successfully extracted from its sources.

However, the yield was not that high. The low % concentration can be added to the egg white and milk, then it will affect the solutions pH and precipitate out the less amounts protein. Another factor is the temperature of the environment where the experiment was conducted. Proteins are easily denatured in hot temperatures. For future experiments, it is recommended that the procedures be done under more suitable room temperatures, in order to avoid protein denunciation and subsequent loss of product, and that the measurements of reagents be done more carefully in order to produce a higher % yield.

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