Rise final research assingment(the one)


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Rise final research assingment(the one)

  1. 1. Isolation, Purification, and Assay of Wheat Germ Phosphatase Wilmarie Morales Natalia Manzano RISE Program May 15, 2012 Abstract:The objective of the experiment was to isolate acid phosphatase from wheat germ and todetermine the protein concentration. The protein was isolated through a centrifuge basedpurification technique and later examined through gel electrophoresis. The protein estimationwas done using the BCA method. The results were not what were expected and must be redonein order to obtain acceptable results. Introduction Acid phosphatase is a type of enzyme that is used to free attached phosphate groups fromThe wheat plants embryo is known as wheat other molecules during digestion. The wheatgerm and it is found in the wheat kernel. The germ embryo uses the phosphatase proteingerm is the most nutritious part of the kernel in growth and germination. It does this bycontaining 28% of protein, which is more catalyzing and hydrolyzing phosphatethan can be found in most meat products. groups from macromolecules and smallerAll the proteins found in wheat germ are molecules that are stored in the wheat seed.different and serve various essentialpurposes for the plant. One of these proteins For this project our goal was to isolate theis the acid phosphatase enzyme. acid phosphatase enzyme from the wheat germ through specific methods of isolation, and to determine the amount of
  2. 2. concentration of said enzyme found in our at 2800 x g at 4oC. The supernatant for thiswheat germ samples. centrifugation was denoted as Fraction I (65 ml). An aliquot of 1.0 ml was frozen and Experimental Procedure saved for later assay of protein concentration Materials and enzymatic activity. Fraction I was then maintained cold andCommercial wheat germ, sold as 1.26 ml of 1.0 M MnCl2 was added to theKretschmer, was used as a convenient solution. The solution was then centrifugedsource for the phosphatase enzyme. The and its supernatant (62 ml) and pellet werereagents used were manganese chloride, collected. The supernatant was namedammonium sulfate, and sodium acetate Fraction II2. The pellet obtained wasbuffer-all at 1.0 M concentration. A suspended in 25 ml of 0.05 M sodiumcommercially purchased BCA kit was used acetate buffer (pH 5.7). When the solutionfor the protein estimation. The stacking and appeared uniform it was centrifuged and therunning polyacrylamide gels were made at supernatant (25 ml) obtained from this10% and 4%, respectively. centrifugation became Fraction III.33.48 ml Methods of ammonium sulfate3 were added to Purification Procedure Fraction II, bringing it to 35% saturation.Twenty five grams of wheat germ were Centrifuge the sample and both the pelletsuspended in 100 ml of deionized water and supernatant (92 ml) are used for further o(4 C). The suspended wheat germ was then purification. The pellet is suspended in 20filtered using cheesecloth, the obtained ml of sodium acetate buffer and centrifuge.filtrate was titled with a volume of 70 ml. The supernatant (21 ml) obtained becomes 1The filtrated was centrifuged for 20 minutes
  3. 3. Fraction IV. Add 51 ml of ammonium 2.5µl of sample. The x100 dilutions weresulfate to Fraction II and raising the solution contained 49.5 µl of 1xPBS for every 0.5 µlto 57% saturation. Place solution in a hot of sample. 8.28 ml of BCA reagent andwater bath until the temperature reaches 165.6 µl of copper sulfate were added to the70oC and imediately place in a cold water samples. The samples were then placed in abath until the temperature lowers to 30oC. spectrophotometer and the absorbance wasOnce the solution has cooled centrifuge and read.the supernatant (136 ml) obtained is denoted A standard curve was created in order toas Fraction V. The pellet obtained from this compare the concentrations of the rest of thecentrifugation is suspended in deionized samples.water and then centrifuged. The obtained 1 All centrifugation was done at 4oC for twentysupernatant (78 ml) becomes our sixth and minutes at 2800xg 3 The addition of ammonium sulfate had to be donefinal fraction (Fraction VI). gradually in order to prevent protein denaturing. Protein Assay SDS-PAGEThe technique chosen to undergo the protein A 10% polyacrylamide gel was prepared.estimation was the Bicinchonic Acid Assay All six fractions were placed in the gels and(BCA). BCA serves the purpose of reacting the gel ran for two hours.with complexes between copper ions andpeptide bonds to produce a purple end Resultsproduct (Stoscheck 2000). Protein EstimationAll six fractions were diluted ten times and After obtaining the absorbance of all ourthen a hundred times. The x10 dilutions samples we were able to calculate ourwere done with 22.5 µl of 1xPBS for every
  4. 4. protein concentrations. For the calculations would reduce. Instead, as shown in Figure B,we eliminated all absorbances that were the concentrations for the second fractionfound to be over 1 and under 0.1(Figure A). were twice as much as the first and then the concentration for Fraction V were nearly six times larger than those for Fraction IV.Figure A- all rows in yellow are the concentrations thatwere removedThis measure was taken because thespectrophotometer reads accurately up to 1.5,any concentration higher than this causes thespectrophotometer to become saturated. Forthis reason we strain our study to a 0.1 to 1 [Figure B] SDSrange in order to obtain more accurate The resulting gel from the SDS-PAGE wasresults. Once the concentrations were unable to show any significant pattern orcalculated the results obtained were not data. What was expected of the gel was topromising. The concentrations calculated show less and less bands as the fractionshad no consistent pattern and no proper were purified. Instead our gel (Figure C)deduction could be done from them. The showed a constant amount of bands for mostexpected was that, as the fractions became gels and then none for others. From this wemore purified the concentration of protein
  5. 5. can determine that our protein isolation was added than the absorbance readings couldunsuccessful. have been affected. These are some of the possible mistakes that could have happened, but it is impossible to know for sure.Discussion The experiment should be redone taking these things into consideration. If the acid phosphatase enzyme is properly isolated than many studies can be conducted afterwards, such as: classification for the phosphatase, determining the concentration of the protein in other specimens and comparing it with the wheat germ’s [Figure C]Our experiment was unsuccessful since the concentration, and also for biomedicalresults obtained were not the ones expected. studies. It is believed that acid phosphataseThis can be accountable to different errors can be used for clinical diagnosis, so, ifthat could possibly have been made during properly isolated, the relationship betweenthe procedure. Most likely the mistakes were this enzyme and human disease can bemade during the stage where ammonium further studied (Bull 2002).sulfate was added to the solution, since this Acknowledgementscan cause denaturing of proteins. Anotherpossible mistake could have been during the To Vibha Bansal PhD and her team ofsample dilution process. If the proper students and tecnitians Edmarie Martinez,volumes of sample and reagents were not
  6. 6. Vivian Rodriguez Cruz, and Alexandra Wheat Germ. European J. Biochem.,Rosado Burgos. 439-44.To the RISE program and its coordinatorsDr. Eneida Díaz, Dr. Elena González, andDr. Robert Ross. References Stoscheck ,2000. CM. Quantitation of Protein. Methods in Enzymology, 50-69. Bull H., Murray P., Thomas D., Nelson P., 2002. Acid phosphatases. Journal of Clinical Pathology, 65-72. Ke-Xue Zhu,Hui-Ming Zhou, and Hai-Feng Qian, 2008. Proteins Extracted from Defatted Wheat Germ: Nutritional and Structural Properties [http://cerealchemistry.aaccnet.org/d oi/abs/10.1094/CC-83-0069] VERJEE Z. H. M., 1969. Isolation of Three Acid Phosphatases from