jove-materia.pdf

Journal of Visualized ECopyright 2012 Creative Commons Attribution-NonCommercial LicensePage 1 of 2Materials List for:Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant RhoProtein(GST-RhoA(G17A)from Epithelial Cell LysatesFaiza Waheed1,2,Pamela Speight1,2,Qinghong Dan1,2,Rafael Garcia-Mata3,Katalin Szaszi1,21Keenan Research Centre,Li Ka Shing Knowledge Institute,St.Michaels Hospital2Department of Surgery,University of Toronto3Department of Cell and Developmental Biology,University of North Carolina at Chapel HillCorrespondence to:Katalin Szaszi at szasziksmh.caURL:http:/ NumberCommentspGEX-RhoA(G17A)AddgeneWill be available soonGenerated in the lab of Dr.KeithBurridge(University of NorthCarolina)(ref 3)DH5Invitrogen18258-012Store-80CSOC mediumBioShop CanadaSOC001.10LBBioShop CanadaLBL407.1Keep sterileGlycerolBioShop CanadaGLY002.1AmpicillinBioShop CanadaAMP201.25Store stock at-20CAgarBioShop CanadaAGR001.500IPTG(Isopropyl B-D-thiogalacto-pyranoside)Calbiochem420322Store stock solution at-20CGlutathione Sepharose beadsGE Healthcare17-0756-01PBS 10 xSigma-AldrichD1408HepesSigma-AldrichH4034NaClBioShop CanadaSOD001.10MgCl2Sigma-AldrichM-9272Add just before useTX-100Sigma-AldrichX100DTTOmniPur,EMD Millipore3860Add just before usePMSFSigma-AldrichP-7626Add just before useProtease Inhibitor 50 xBD Biosciences51-21426ZAdd just before useComplete Mini,EDTA-free 10 xRoche Group11 836 170 001Add just before useLaemmli Sample Buffer 2xBio-Rad161-0737-mercaptthanolSigma-AldrichM7154Add just before useCoomassie Brilliant BlueBio-RadR-250Acetic AcidCALEDON1000-1MethanolCALEDON6701-7Bio-Rad Protein AssayBio-Rad500-0114BLUeye ladderFroggaBioPM008-0500Table 1.Reagents usedRC-5B centrifugeSorvall,Thermo ScientificSS-34 RotorUse at 4CCentrifugeSorvall,Thermo ScientificST-16RUse at 4CMicro centrifugeEppendorf5417RUse at 4CBacterial shakerINFORS HT EcotronAG CH-1403 BottmingenUse at 37C or at 22CJournal of Visualized ECopyright 2012 Creative Commons Attribution-NonCommercial LicensePage 2 of 2SonicatorBransonSonifier-450Use at RTRotatorGlas-Col099A CR4012Use at 4CTable 2.Specific equipments used