+Analysis andIsolation ofPlasminogenActivator fromMammalian CellCulture BrothDelaine Zayas-Bazán BurgosJohn Muñoz TorresVi...
+IntroductionProteins are macromoleculescomposed of amino acids, joined bypeptide bonds.Our purpose was to isolatePlasmi...
+Introduction“Magnetic nanoparticles continue to garnerwidespread interest in biomedicalapplications,such as visualizatio...
+IntroductionWhat are nanoparticles? PABA Treated Magnetic Nanoparticles
+HypothesisPlasminogen Activator can besuccessfully isolated from amammalian cell culture utilizing PABAtreated Magnetic ...
+Work Plan• MNP• ChromatogramSeparation• SDS Page• Zymography• ProteinEstimation• EnzymeActivityAnalysis
+SeparationSeparationImage from Dr. Bansal’sHandout
+MaterialsReagents: HeLa Broth Binding Buffer Elution Buffer Regeneration Buffer PABA-Epoxy-IOMNPs (50.0mg)Equipmen...
+Materials Seven (1.5mL) centrifugetubes One 15.0 mL centrifugetube One magnet Ice Bucket with ice Test Tube rack 10...
+Analisis
+Materials needed for SDS-Page Stacking gel 4.0% dH2O 2.795 ml Stacking buffer 1.25 ml Acrylamide 0.662 ml 20% SDS 25...
+Materials needed for zymography Separating Gel 10% dH2O 1.788 ml Separating buffer 1.67 µl Acrylamide 2.22 µl 20% SD...
+ Separates proteins according to their electrophoreticmobility and no otherphysical feature. SDS is a detergent applied...
+Procedures
+• SDS-Page• ZymographyPolymerize• Load and run thegel• Leave overnight atroom temperaturein fixativeSDS-Page• Load and ru...
+Results0.0000.5001.0001.5002.0002.5003.0000 2 4 6 8 10 12 14 16 18AbsotbanceVolumeChromatogramEluateWashLoad
+Results These results showsthat the protein ofinterest, PA, wassuccessfully isolatedfrom the bulk ofproteins available ...
+Resultsy = 1177.9x - 5.0193R² = 0.94780.0050.00100.00150.00200.00250.00300.00350.000.000 0.050 0.100 0.150 0.200 0.250 0....
+ResultsLadderHeLaLoadHeLaSpent EluatePlasminogenActivatorSDS-Page with SilverStaining (WithoutProtein Estimation)LadderHe...
+ConclusionsThe experiment was successfulPlasminogen Activator was isolated andanalyzed.The protein was isolated at a f...
+Further WorksThis experiment can be utilized in furtheranalysis of the protein PlasminogenActivatorThe experiment can a...
+References Balaure P, Buteica A, Grumezescu A, Mihaiescu D, Mihaiescu O,Mogosanu D,Trăistaru V,Vasile B. 2012.Bioassay a...
+References Erf Gisela F, Kanayeva Damira A, Li Yanbin, Rhoads Douglas, SlavikMichael F, Tung Steve, Wang Ronghui. 2012. ...
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Rise vibha investigation presentation (1)

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Rise vibha investigation presentation (1)

  1. 1. +Analysis andIsolation ofPlasminogenActivator fromMammalian CellCulture BrothDelaine Zayas-Bazán BurgosJohn Muñoz TorresVibha Bansal PhDMr. Javier RosadoMr. Carlos CastrodadMrs. Natalia Espada
  2. 2. +IntroductionProteins are macromoleculescomposed of amino acids, joined bypeptide bonds.Our purpose was to isolatePlasminogen Activator from amammalian cell culture HeLa Broth
  3. 3. +Introduction“Magnetic nanoparticles continue to garnerwidespread interest in biomedicalapplications,such as visualization agents inMRI, therapeutic vehicles for drug deliveryand heat mediators in hyperthermia.”Reynolds and Hilt (2010).Mihaiescu and his team (2012) workedwith nanoparticles as therapeutic vehiclesfor drug delivery in E.coli
  4. 4. +IntroductionWhat are nanoparticles? PABA Treated Magnetic Nanoparticles
  5. 5. +HypothesisPlasminogen Activator can besuccessfully isolated from amammalian cell culture utilizing PABAtreated Magnetic Nanoparticles
  6. 6. +Work Plan• MNP• ChromatogramSeparation• SDS Page• Zymography• ProteinEstimation• EnzymeActivityAnalysis
  7. 7. +SeparationSeparationImage from Dr. Bansal’sHandout
  8. 8. +MaterialsReagents: HeLa Broth Binding Buffer Elution Buffer Regeneration Buffer PABA-Epoxy-IOMNPs (50.0mg)Equipment: Sonicator Rocker (120 rpm) UVSpectrophotometer
  9. 9. +Materials Seven (1.5mL) centrifugetubes One 15.0 mL centrifugetube One magnet Ice Bucket with ice Test Tube rack 100-1,000 μL Micropipetteand tips Stop clock Beaker for waste collection Labeling Tape Marker Gloves
  10. 10. +Analisis
  11. 11. +Materials needed for SDS-Page Stacking gel 4.0% dH2O 2.795 ml Stacking buffer 1.25 ml Acrylamide 0.662 ml 20% SDS 25.0 µl 10% Glycerol 25.0 µl TEMED 6.25 µl APS (100 mg/ml) 25.0 µl Separation gel 10.0% dH2O 1.68 ml Separating buffer 1.67 ml Acrylamide 2.22 ml 20% SDS 50.0 µl 10% Glycerol 100.0 µl TEMED 1.67 µl APS (100mg/ml) 50.0 µl
  12. 12. +Materials needed for zymography Separating Gel 10% dH2O 1.788 ml Separating buffer 1.67 µl Acrylamide 2.22 µl 20% SDS 50.0 µl 10% Glycerol 100.0 µl Fibrinogen 50.0 µl Plasminogen 40.0 µl TEMED 1.67 µl Thrombin 2.0 µl Amm.Per sulfate 50.0 µl Stacking Gel 4% dH2O 2.975 ml Stacking buffer 1.25 ml Acrylamide 0.662 ml 20% SDS 25.0 µl 10% Glycerol 25.0 µl TEMED 0.25 µl Amm.Per sulfate(100mg/mL) 25 .0 µl
  13. 13. + Separates proteins according to their electrophoreticmobility and no otherphysical feature. SDS is a detergent appliedto protein sample to imparta negative charge to all ofproteins. In most proteins, the binding ofSDS to the polypeptide chainimparts an even distribution ofcharge This causes a separation byapproximate size duringelectrophoresis. Based on SDS-PAGE Includes a substrate co-polymerized with the gel Utilized for the detectionof enzyme activity. Samples are prepared in thestandard SDS-PAGE treatment buffer WITHOUT a reducing agent or boilingin order for the enzyme to retain itsnative state It is incubated at 37°C to leave theenzymes the correct environment forthem to digest the protein or substrate. This causes white bands, in which theprotein has been digested.SDS-Page Zymography
  14. 14. +Procedures
  15. 15. +• SDS-Page• ZymographyPolymerize• Load and run thegel• Leave overnight atroom temperaturein fixativeSDS-Page• Load and run thegel at 4°C• Stain with CoomasieBlue• Incubate overnightat 37°C• DestainZymo• Take pictures• Perform ProteinEstimation• Perform EnzymeActivityAnalysis
  16. 16. +Results0.0000.5001.0001.5002.0002.5003.0000 2 4 6 8 10 12 14 16 18AbsotbanceVolumeChromatogramEluateWashLoad
  17. 17. +Results These results showsthat the protein ofinterest, PA, wassuccessfully isolatedfrom the bulk ofproteins available They also provide anascertain that is ourprotein, and not anyother protein.HeLaLoadHeLaSpentEluatePlasminogenActivatorZymography withCoomassie BlueStaining
  18. 18. +Resultsy = 1177.9x - 5.0193R² = 0.94780.0050.00100.00150.00200.00250.00300.00350.000.000 0.050 0.100 0.150 0.200 0.250 0.300Enzymeactivity(IU/mL)Abs at 405 nmEnzyme Activityy = 1.4221x - 0.0141R² = 0.993-0.2000.0000.2000.4000.6000.8001.0001.2000.000 0.200 0.400 0.600 0.800AmountofProteinAbsorbancy at 562nmProtein Estimation
  19. 19. +ResultsLadderHeLaLoadHeLaSpent EluatePlasminogenActivatorSDS-Page with SilverStaining (WithoutProtein Estimation)LadderHeLaLoadHeLaSpentEluatePlasminogenActivatorKeratinSDS-Page with SilverStaining (With ProteinEstimation)
  20. 20. +ConclusionsThe experiment was successfulPlasminogen Activator was isolated andanalyzed.The protein was isolated at a foldpurification of 10 The fold purification is a measure of how muchmore pure your protein is after a purification stepin comparison to the crude.
  21. 21. +Further WorksThis experiment can be utilized in furtheranalysis of the protein PlasminogenActivatorThe experiment can also be used as a basefor further experimentations about proteinseparation with treated mageneticnanoparticles
  22. 22. +References Balaure P, Buteica A, Grumezescu A, Mihaiescu D, Mihaiescu O,Mogosanu D,Trăistaru V,Vasile B. 2012.Bioassay andElectrochemical Evaluation Of Controlled Release Behavior OfCephalosporins From Magnetic NanoparticlesDigest Journal ofNanomaterials and Biostructures. [Internet]; [Revised 2012February 24, Cited 2013 May 16] 7(1). <ahref="http://search.ebscohost.com/login.aspx?direct=true&db=a9h&AN=74616350&site=ehost-live">BIOASSAY ANDELECTROCHEMICAL EVALUATION OF CONTROLLED RELEASEBEHAVIOR OF CEPHALOSPORINS FROM MAGNETICNANOPARTICLES.</a Eisenhour David, Hickman Jr. Cleveland, I’Anson Helen, Keen SusanL, Larson Allan, Roberts Larry S. 2008. Integrated Principals ofZoology. Fourteenth Edition. United States of America. McGraw-Hill.
  23. 23. +References Erf Gisela F, Kanayeva Damira A, Li Yanbin, Rhoads Douglas, SlavikMichael F, Tung Steve, Wang Ronghui. 2012. Efficient Separation andSensitive Detection of Listeria monocytogenes Using an ImpedanceImmunosensor Based on Magnetic Nanoparticles, a Microfluidic Chip, andan Interdigitated Microelectrode. Journal of Food Protection. [Internet];[Revised 2012 November; Cited 2013 May 11] 75(11).http://search.proquest.com.uprcdb.cayey.upr.edu:2048/docview/1115592225?accountid=44829 Frimpong Reynolds A, Hilt J Zach. 2010.Magnetic nanoparticles inbiomedicine: synthesis, functionalization and applications. Magneticnanoparticles in biomedicine: synthesis, functionalization and applications.Future Medicine Ltd [Internet]; [Revised 2010 November; Cited 2013 May11] 5(9) DOI:http://dx.doi.org.uprcdb.cayey.upr.edu:2048/10.2217/nnm.10.114http://search.proquest.com.uprcdb.cayey.upr.edu:2048/docview/815858529?accountid=44829

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