BacterialProteinExtraction-miniscale-Sonication

ProteinExtraction

1)ResUSPendpelletof10mlcellculturein1mllysisbuffer(or100mlbacterialcultureforverylowexpressionlevel).

SuggestedLysisbuffer:140mMNaCl;2.7mMKCL;10mMNa₂HPO₄;1.8mMKH₂PO_4;pH7.3(PBS)or100mMNaCl;25mMTrisHCl;pH8.0optional0.02%NaN₃(azide)optionalproteaseinhibitors

Optionaladditivestothelysisbuffera)1mMPMSForproteaseinhibitorcocktail1:200(cocktailforbacterialcells#P-8849fromSigma)b)Dnase100U/mlor25-50ug/ml(SIGMADN-25).Incubate10min4°Cinthepresenceof10mMMgCl₂.c)Lysozime0.2mg/ml.Incubate10min4°C.d)ßME,DTTorDTEupto10mMforproteinswithmanycysteines.e)0.1-2%TritonX-100,NP40;oranyotherdetergentthatdonotaffecttheBIOLOGicalactivityofyourprotein.f)10%glycerol(forstABIlizationoftheproteinandpreventionofaggregation).

2)Sonicateinicebucket3x10secormoreifthecellsarenotcompletelydisrupted(Lysisiscompletewhenthecloudycellsuspensionbecomestranslucent.Avoidproteindenaturationbyfrothing).

3)Spin5min13000rpm4°C.Separatesolubleproteins(supernatant)frominsolubleorinclusionbodiesproteins(pellet).Usesupernatantfornextstep.Keepsampleof40ulofsupernatantforPAGE-SDSandWesternblot:solubleproteins

4)Resuspendpelletinanother1mllysisbufferandkeepsampleof40ulforPAGE-SDSandWesternblot:insolubleproteins,orunlysedcells.

Analysisofresults-Troubleshooting

Expectover-expressedproteintobefoundonlyinthecrudesupernatant.Ifmostoftheproteinremainsinsolubleafterextraction,trya)TochangelysisbufferbyaddingßME,DTT,glycerol,detergentsormoreNaCl.Ifonlypartofitisinsoluble,b)Orre-extractpelletwithmorebuffer,c)Orusemorelysisbufferduringextraction,d)Orperformamoreintensivesonication,e)Orincubatewithlysozymebeforesonication.f)Ortrythedenaturatingprotocolextraction(4-6Mguanidine-HCl;4-8Murea;alkalinepH>9.0;0.5-2%TritonX-100;0.5-2%N-lauroylsarcosineorotherdetergents)g)SeeSolubilityStudies:Preparationofsoluble/insolubleproteinfromcellsaccordingtothePROTEINEXPRESSIONANDPURIFICATIONFACILITYoftheEUROPEANMOLECULARBIOLOGYLABORATORY

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