In this experiment, albumin and casein were extracted from egg white and milk, respectively due to the

abundance of the target proteins from the said sources. The purification procedure takes into account the physical and chemical properties of the protein such as solubility, polarity and temperature needed to obtain maximum recovery. Suitable purification methods are determined by the activity of the target protein to different solvents and treatments, and the contaminating compounds present together with the target protein. 1 Purification started by the disruption of the cell membrane of the source through homogeni ation to isolate the target proteins. !omogeni ation by mechanical disruption as in the case of egg white was done by stirring and pressing of the egg white during filtration. " #nce the membranes were disrupted, the target proteins were isolated by centrifugation and fractional precipitation. $entrifugation is the process in which particles in a solution is separated accordingly depending on si e, shape and density. In this experiment, centrifugation was done to separate the target proteins, nucleic acids and other unbroken cells from the broken ones. #n the other hand, fractional precipitation involves the separation of particles from the solution depending on its solubility. In this experiment, fractional precipitation may be observed when p! was lowered, organic solvents and neutral salts were used and when there was a need for ice bath and refrigeration. Isoelectric precipitation is the ad%ustment of the p! of a solution as to reach its isoelectric point, as may be seen in the extraction of casein in milk using !$l. Proteins, having both positive and negative charges due to the amino and carboxyl groups present may then have a neutral net charge. Isoelectric point is the point in which the net charge of the protein is e&uivalent to ero. 't this point, because of the absence of a charge, a decrease in the protein(water molecule interaction causing for the solubility to be lowered will result to an increase in protein(protein molecule interaction thereby forming a precipitate. The same concept may be observed when organic solvents miscible with water, such as ethanol and acetone, were used. Such solvents have a low dielectric constant which further lowers that of the water which decreases its solvating power by increasing exposing more hydrophobic sites that promotes hydrophobic interactions. ) Proteins also have different solubility when neutral salts are added. They tend to be insoluble to concentrated salt solutions, as may be seen when saturated *+! ,-"S#, was used in the extraction of albumin from egg whites. This is because of the fact that when the concentrated salt solution was added, the ions tend to attract the water molecules blocking the hydrophobic sites of the protein. #nce these sites are exposed, protein molecules interact with one another causing in aggregation and therefore forming insoluble precipitates. This is called the salting out effect. " #n the other hand, when dilute concentrations of salt was used as in the use of ...1 / +a#! and ..01 +a$l, proteins tend to be soluble because of the weakening of the attractive forces between proteins, called salting in effect. 2astly, proteins, when exposed to high temperatures, denature which disrupts the natural conformation of the proteins affecting its function. Therefore, to avoid denaturation, the process should be done at lower working temperatures, and refrigeration was needed for the isolated proteins. !owever, in plants and microorganisms, simple mechanical disruption of cell membrane is not enough due to the presence of cell wall and other compounds such as phenols and proteinases which protects further the contents of the cell and decreases the activity of the target protein. To address these problems, en ymatic lysis using lyso yme and extraction buffers is applied to digest and stabili e the membrane. +evertheless, certain part of plants that are rich in specific en ymes and thus, used primarily for extraction such as the seed, tuber and tap roots also known as storage organs. #n the other

hand, a direct approach is applied to animal tissues due to weak membrane holding the target proteins. The purification method for animal tissues then depends on source, whether it is a soft or hard muscle, and to the complexity of the target protein, extracellular matrix or membrane bound. 3xtracellular matrix proteins re&uire more extensive extraction with the use of chemical hydrolysis and4or proteolytic cleavage because of low solubility than the membrane bound. ,, 5 'fter extraction, the proteins were sub%ected to &uantitation through spectrophotometry, employing three different methods6 7arburg($hristian 'ssay, 8radford 'ssay and 8iuret 'ssay. 7arburg($hristian 'ssay involves the direct measurement of 9: absorbance of amino acids such as Tyrosine and Tryptophan at ";. nm since most proteins also exhibit absorbance at the said wavelength. !owever, since proteins may contain different amount of amino acids, this method is not suitable for &uantitative determination of concentrations of protein mixture. 3ven though, this techni&ue, being non destructive, fast and easy, also provides correction for contaminants such as nucleic acids which commonly absorbs at "<. nm. #n the other hand, both the 8radford and 8iuret 'ssay are colorimetric methods which employ the binding of $oomassie 8rilliant 8lue dye and $u "= respectively, to the protein. 7hile the 8radford 'ssay causes a shift in the absorption wavelength of the dye from ,<5 to 505 nm due to its binding with the protein, the 8iuret 'ssay forms a violet solution due to the complexation of the protein and $u"= with absorbance that can be measured at 5,. nm. < >I+S3?T !3?3@ #ther methods for protein &uantification include the 2owry method and 8icinchoninic acid *8$'method, both colorimetric methods takes advantage of the reducing power of amino acids such as Tyrosine and Tryptophan, at alkaline solutions to reduce $u "= to $u= which would later bind to the reagent. In 2owry method, the $u= binds with the Aolin( $iocalteu reagent which is a reduced phosphomolybdate(phosphotungstate solution to produce a blue color solution to be measured at <<. nm. This process, however, destroys the target protein. /eanwhile, in 8$' 'ssay, the $u = binds with the 8$' to produce a violet colored solution measured at 5<" nm. Though the color yield varies between proteins due to the number of reducing amino acids, this method is useful in determining the changes in protein content as it can be used in varying temperatures. This method should be accompanied with the use of buffer to eliminate the error brought about by non protein reducing compounds. ) /ost protein &uantification methods are neither straightforward nor exact due to the variations in the properties of target protein. /ethods present cannot address fully each problem such as the presence and amount of amino acids in each protein, and the presence of contaminating compounds that has not been full separated from the target protein. :ariations in the result may be due to personal errors in the preparation of solutions used in the experiment and in the preparation of the sample during the use of the spectrophotometer, as fingerprints or bubbles may be present in the cuvette, blocking the path of the light. Bifferences as well in the degree of mechanical disruption *stirring etc- may cause the lower values of extracted proteins.

?3A3?3+$3S6 >1@ Canson !.$. D ?yden 2. 100;. Protein Purification 2nd Edition. Cohn 7iley D Son, Inc. $anada. pp.5(E >"@ $ampbell, /.F. D Aarrell, S. #. ".1". Biochemistry 7th Edition. 8rooks4$ole, $engage 2earning. 9S'. pp.11E(11; >)@ Swit er, ?. 2. D Garrity, 2. A. 1000. Experimental Biochemistry. 7.!. Areeman and $ompany. 9S'. pp.01(0, >,@ Aido, ?. C. et al. "..,. Protein Extraction From Plant Tissues, Methods in Molecular Biology, vol.2 ! Protein Purification Protocols 2nd Edition Ed "y P. #utler. !umana Press Inc. 9S'. >5@ Shekel, C. /. "..,. Preparation of Extracts From $nimal Tissues, Methods in Molecular Biology, vol.2 ! Protein Purification Protocols 2nd Edition Ed "y P. #utler. !umana Press Inc. 9S'. ><@ ".1). Biochemistry %a"oratory Manual. 8iochemistry 'cademic Group, Institute of $hemistry, 9P Biliman. Philippines >E@ Protein $nalysis& 'etermination of Protein #oncentration . (http!))pu"lic.callutheran.edu)*revie)"iochemistry)Protein&analysis&la".pdf+ >;@ "..0. #omparison of Protein $ssays. http!)),,,.col"y.edu)chemistry)#-./7)la"oratory)expt2.pdfI ?etrieved ?etrieved from from H

>0@ Sattayasai, +. Protein Purification. ?etrieved from (http!))cdn.intechopen.com)pdfs)207/1)2nTech& Protein3purification.pdf+