Isolation and fractionation of proteins from skim milkDocument Transcript
Isolation and fractionation of Proteinsfrom skim milk (Casein)Luis A. Pérez Soto1, Osvaldo J. Vega Rodríguez1, Alexandra Rosado Burgos2, Marienid FloresColón2, Vivian Rodríguez Cruz2, Vibha Bansal Ph.D2 1 2Department of Biology , Department of Chemistry , University of Puerto Rico at Cayey, RISE Program____________________________________________________________________________Abstract Since the existence the human has consumed milk and know is recommendedbecause of the high levels of calcium, but it also a good source of proteins. Thesephosphoproteins are called casein and compose 80% of the proteins in the milk. Herewe will extract this protein from skim milk and identify which are the most abundanceones in the milk. We will apply the techniques of protein estimation and SDS-page gelto extract the protein and quantify how much proteins we have in a sample. By themolecular weight we will identify the casein proteins in the milk.______________________________________________________________________Introduction Casein is the major protein in milk and the most commons are the alpha, beta,gamma and kappa. The alpha and beta casein are phosphoproteins with a phosphatebeing present mainly in the form of O-phosphorine residues. It is found as a suspensionof particles called casein micelles. The isoelectric point of casein is 4.6, but becausemilk has a pH of 6.6 the casein has a negative charge. Adding the acid to have less pHmakes the negatives charges on the outer surface of the micelle (solubilice unit) to
neutralize and the neutral protein precipitates. Casein is used for paints, glue, cheesemaking, plastics and fiber, protein supplements, and medical and dental uses. Thecasein is a mixture of at least four proteins, and differs primarily in the molecular weightand the amount of phosphate groups.______________________________________________________________________MATERIAL & METHODS To extract the casein protein from the milk we mix 100ml of skim milk with 300mlof tap water using a magnetic stirrer. Because milk is base we added (10ml) of 10%HCL so we can have a pH of 4.8. After stirring for 10 minutes we let the content settleand then centrifuge for 15 minutes at 4,000 rpm. To clear the sample we suspendedthe precipitate in 200ml of deionized water (dH2O) letting the sample settle anddecanting the supernatant. After decanting two times we suspended the precipitateonce more in 200mL of dH2O and collect it on a Buchner funnel using three thicknessesof filter paper to filter the solution and separate the precipitate for the supernatant by theuse of a pump and free the solution of chloride ions. We repeated this process fourtimes until the silver nitrate test proves that we didn’t have any chloride ions. The use ofsilver nitrate test is easy just pour some drops into an aliquot of the supernatant. Whenclearing of the chloride ions we suspended the casein in 20mL of 95% of ethanol andfiltrate it again in the funnel and after we filtrated with acetone to dry the precipitate andbeing able to work with. (Stenesh, 1984)Protein Estimation:
Weight different amount of casein in duplicates and pour in a 15mL centrifugetubes. We measure .001g, .007g, .010g, and .015g and suspended them in 1,000mL of1x PBS. Also added 2mL of Bicinchoninic acid reagent (BCA) with copper solution toone set and to the other 2mL of dH2O; the sets with water were use as negativecontrols. Then prepare the calibration curve in the microplate using BSA standardprotein (1mg/mL). We incubated the tubes and microplate for 30 minutes and after justcentrifuged the tubes at 4,000rpm for 15min. To the wells (not the ones with thestandards) is needed to add 241.5uL of the precipitate and wait for dilution (theorganization of the wells can be seen in Table 1). At last we read the absorbance levelsin the microplate reader.RESULTSProtein Estimation Table #1: Protein estimation concentrationsUsing the formula obtained in Graph 1 we obtained the concentrations of proteins ineach one of the samples. This formula was obtained from the standard curve draw
using the five standards of the experiment. The concentrations show in Table 1 wereaccurate and show a significant different between the sample, we know that our resultsare reliable because the formula used to calculate the concentrations have a square Rof .98. Other data that tells us that the concentrations are correct is that the higher theabsorbance the higher the sample quantity and concentrations of proteins. This wasdone to prove that we correctly have isolated the protein from the milk. Graph #1: Standard Curve of absorbance levels Standard Curve 0.7 0.6 y = 0.52x + 0.097 0.5 R² = 0.981 Absorbance 0.4 0.3 Series1 0.2 Linear (Series1) 0.1 0 0 0.2 0.4 0.6 0.8 1 1.2 ConcentrationSDS-PAGE GelsIn the first pair of gels the bands of the skim milk were correct and show almost nodifference between them. The problem was that the marker didn’t express well so wedidn’t have something to determine the molecular weight of the proteins, also the controlcasein didn’t express so we didn’t have anything to compare our result, and our caseinwasn’t good in either concentrations.
SDS Page Gel #1 SDS Page Gel #2Gel #3 give us good result in expressing the marker, skim milk and our casein but theproblems was that the control casein wasn’t diluted and it show a big band that invadepart of our casein area. To solve this problem we make a fourth gel that gives us theperfect results (Gel #4). In gel #4 the bands of the skim milk, control casein and theexperimental casein expressed in the same places. By analyzing this last gel and by thehelp of the ladder created by the marker we determine the molecular weight of thebands and find that the bands belonged to the alpha, beta, gamma, and kappa casein. SDS Page Gel #3 SDS Page Gel #4
Conclusion and Future WorksThere may be other caseins but for the purpose of this project we isolated the casein inthe skim milk and were able to found that this four are the more abundant in skim milk.The more abundant is the alpha casein that can be αS1 or αS2, the second abundancelevel is the beta casein follow by gamma casein and at last the kappa casein, all of thiswas concluded by analyzing the SDS-page gel. This complex of caseins forms the 80%of proteins in the cow and 40% of proteins in human milk. These phosphoproteinsbeing so abundance in milk and its derivates is needed to be study more deeply, weintent to develop techniques that can isolate a specific casein protein to later analyzetheir functions.AcknowledgmentThis project wouldn’t have been done without the leading of the student mentors:Alexandra Rosado Burgos, Marienid Flores Colón, and Vivian Rodriguez Cruz. Thecollaboration and guidance of Vibha Bansal Ph.D. by letting us work in her laboratory. Itwas supported by the RISE Program.References Stenesh J. Experimental Biochemistry. Kalamazoo (MI): Western Michigan University; 1984. 87 p.
R.K. Dewan, A. Chudgar, R. Mead, V.A. Bloomfield, C.V. Morr. 1974.Molecularweight and size distribution of bovine milk casein micelles [Internet;.[2012 May09] http://dx.doi.org/10.1016/0005-2795(74)90086-5