Yolanda rodriguez 28nov2011


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Yolanda rodriguez 28nov2011

  1. 1. Extraction and Quantification ofProteins in Syzygium jambos used asDiabetic Adjuvants in Puerto RicoYolanda I. Rodríguez-RiveraBIOL 4990Mentor: Dr. Jannette Gavillán-Suárez
  2. 2. Proteins• Proteins arebiochemicalcompoundsconsisting ofpolypeptides thatfacilitate a biologicalfunction.Protein FunctionsRamírez Ramírez, G, Heil, M, Orona Tamayo, D, Adame ÁlvarezRM, Ramírez Ramírez, DT, Durán Flores, FD. 2008. ANÁLISIS DEPROTEÍNAS EN GRÁNULOS Y HOJAS DE Acacia hindsii Y ENHORMIGAS DE LA REGIÓN DE IRAPUATO . Guanajuato (MX):Universidad Autónoma de Querétaro (MX). Disponible en:http://www.uaq.mx/investigacion/difusion/veranos/memorias-2008/10VeranoRegionCentro/17UAQRamirezRamirez.pdf
  3. 3. Solubility of Proteins Hydrophilic and Hydrophobic residueson surfaceCharged and polar surfaces will havehigher solubility
  4. 4. Previous studiesA study shows that a protein named ADMc1 inthe seeds of Momordica charantia(cundiamor) has hypoglycemic/anti-hyperglycemic properties.United States Patent Application Publication, Antidiabetic Protein: Pub.No: US2011/0046054
  5. 5. Previous studies• ADMc1 was found highly effective and neededto be administered only once a day tomaintain normal blood glucose levels.• The protein was detected and purified fromthe seeds of the fruit the plant provides.• Published work states that the polypeptide-pis made of 166 amino acid residues and saidto be of 11 kDa.
  6. 6. Specific aims of this studyTo extract proteins on leaves of Syszygiumjambos (Pomarrosa del río). To quantify the proteins by the Lowrymethod.
  7. 7. RelevanceDetermination of proteins that could serve asbiomarkers of hypoglycemic/anti-hyperglycemic activities for the reduction ofblood glucose in diabetic patients.
  8. 8. Methodology : Extraction of Proteins3 replicatesof the plantWash pellet withacetone, 10% TCAand 80% acetone.Centrifuge.2 g of of drypellet isresuspended inPhenolAdd dense SDSbuffer andcentrifugeSeparate phases andprecipitate Ph phasewith methanol andammonium acetate.Recoverinsolubleproteins bycentrifugationWash 3 timeswith methanolicammoniumacetate.Wash with 80%acetoneQuantifyproteins inpellet andsupernatant
  9. 9. Methodology: Protein QuantificationBSAConcentrationand CalibrationCurveAdd BSA [0, 20, 40,60, 80, 100 µl] tothese tubes & 100µl of unknownsAdd 2 ml of LowrySolution to eachone of the testtubesIncubate at roomtemperature for 30minutesAdd 0.2 ml ofdiluted Folin-phenol solution toeach tubeIncubate 10minutes at roomtemperatureVortex each tubeimmediatelyDetermineabsorbance of eachsample at 600 nmPlot absorbance vsmg BSA to obtain acalibration curve
  10. 10. Lowry colorimetric reaction
  11. 11. Resultsy = 7.7027x + 0.0333R² = 0.993900. 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04Absorbance(600nm)BSA Concentration (mg/ml)BSA Calibration Curve
  12. 12. Results for Syszygium jambos supernatant• Concentrations of unknowns:– X1 = 0.33 mg/ml– X2 = 0.57 mg/ml– X3 = 0.44 mg/ml[Protein] = 0.45 mg/ml of supernatant[Protein] = 0.23 mg/g dry weightIn comparison with:[Protein] = 0.25 mg/g dry weight(Gómez-Vidal, et.al)
  13. 13. Analysis of Results• The amount of protein found in this assay iscomparable to results in Gómez-Vidal, S, et.al.• Given that they were found in the supernatant, theycould be protein inside the cell.• The proteins found on the pellet, which areinsoluble, are probably structural and could pertainto the cell wall.
  14. 14. Future Work• Analyze protein in leaves of studied plantsand categorize them by their function via 2-dimensional electrophoresis.• Extract and quantify proteins in leaves ofTapeinochious annasae, Costus speciousus andTradescantia spathacea.
  15. 15. ReferencesGómez-Vidal S, Tena M, López-Llorca LV, Salinas J (2007). Protein Extraction fromPhoenix dactylifera L. leaves, a recalcitrant material, for two dimensionalelectrophoresis. Electrophoresis, 29: 448-456.Hurkman WJ, Tanaka CK (1986). Solubilization of Plant Membrane Proteins forAnalysis by Two-Dimensional Gel Electrophoresis. Plant Physiol., 81: 802-806.Biology at Davidson [Internet]. 2000. Biology at Davidson in North Carolina. ProteinDetermination - Lowry Procedure; 2011 Sep 16. Available from:http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.htmlRamírez Ramírez, G, Heil, M, Orona Tamayo, D, Adame Álvarez RM, RamírezRamírez, DT, Durán Flores, FD [Internet]. 2008. ANÁLISIS DE PROTEÍNAS ENGRÁNULOS Y HOJAS DE Acacia hindsii Y EN HORMIGAS DE LA REGIÓN DEIRAPUATO . Guanajuato (MX): Universidad Autónoma de Querétaro (MX).Disponible en: http://www.uaq.mx/investigacion/difusion/veranos/memorias-2008/10VeranoRegionCentro/17UAQRamirezRamirez.pdf
  16. 16. Extraction and Quantification ofProteins in Syzygium jambos used asDiabetic Adjuvants in Puerto RicoYolanda I. Rodríguez-RiveraBIOL 4990Mentor: Dr. Jannette Gavillán-Suárez