+Analysis andIsolation ofPlasminogenActivator fromMammalian CellCulture BrothDelaine Zayas-Bazán BurgosJohn Muñoz TorresVibha Bansal PhDMr. Javier RosadoMr. Carlos CastrodadMrs. Natalia Espada
+IntroductionProteins are macromoleculescomposed of amino acids, joined bypeptide bonds.Our purpose was to isolatePlasminogen Activator from amammalian cell culture HeLa Broth
+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
+IntroductionWhat are nanoparticles? PABA Treated Magnetic Nanoparticles
+HypothesisPlasminogen Activator can besuccessfully isolated from amammalian cell culture utilizing PABAtreated Magnetic Nanoparticles
+ 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
+• 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
+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
+ResultsLadderHeLaLoadHeLaSpent EluatePlasminogenActivatorSDS-Page with SilverStaining (WithoutProtein Estimation)LadderHeLaLoadHeLaSpentEluatePlasminogenActivatorKeratinSDS-Page with SilverStaining (With ProteinEstimation)
+ConclusionsThe experiment was successfulPlasminogen 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.
+Further WorksThis experiment can be utilized in furtheranalysis of the protein PlasminogenActivatorThe experiment can also be used as a basefor further experimentations about proteinseparation with treated mageneticnanoparticles
+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.
+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