BacterialProteinExtraction(mini-scale)UsingB-Per

B-Per(Pierce,Catnumber:78248)Extractionof1.5mlbact.cultureOD600nm1.5-3.5

Ausefulprocedureforinitialscreeningofexpressionconditionsinsteadofsonicationofbacteria;notrecommendedforlargescalepreparationofprotein.NotalwaystheproteinactivityisconservedafterB-Perextraction.

1.Spinbacterialcells10min5000rpminamicrocentrifuge4°C(cellscaneitherbeusedfreshorfrozenat-70°C).

2.ResUSPendcellsin300µlofB-Perreagent(Pierce)byeithervigorousvortexingorbypipettingupanddownuntilthecellsuspensionishomogenous.Vortexfor1moremin.(ifsuspensionistooviscousaddDnase100U/mlor25-50µg/ml(SIGMADN-25).Incubate10min4°Cinthepresenceof10mMMgCl₂

3.Spin13000rpm5min4°C,toseparatesolubleproteinsfromtheinsolubleproteinsinthepellet.Collectsupernatant.Keep40µlsampleofsupernatantforPAGE-SDSorwesternblot:solubleproteins.

4.Resuspendpelletin300µllysisbuffer(25mMTrisHClpH8.0;0.1MNaCl;10%glycerol;0.1%TritonX-100and1mMPMSF).Spin13000rpm5min4°C,toseparatedetergent-solubleproteinsfrominclusionbodiesinthepellet.Collectsupernatant.Keep40ulsampleofsupernatantforPAGE-SDSorwesternblot:detergent-solubleproteins.

5.Forinclusionbodiespurification,add(lysozime6µlof10mg/ml)totheresuspendedpellettoaf.c.of200µg/ml,vortex1min.Add1mlofB-Per1:10tothesuspensionandvortexforanother1min.Collectinclusionbodiesbycentrifugation13000rpm10min4°C.Resuspendpelletin1mlofB-Per1:10.Spinandrepeatextractionagain.PelletcanberesuspendedinsamplebufferforPAGE-SDS:inclusionbodiesfractionorresuspendedindenaturantbufferofUreaorGuanidine-HCl(seenextpoint).

6.Forinclusionbodiesresuspensionpreparelysisbuffer(withoutdetergent)containing8Mureaor6MGuanidine-HCl.Resuspendcellsin300µlbuffer+UreaorGuanidine-HClbypipettingupanddownuntilthecellsuspensionishomogenous.Spin13000rpm5min4°C.Collectsupernatant.Keep40µlsampleofsupernatantforPAGE-SDSorwesternblot:inclusionbodiesproteins.PrecipitateGuanidine-HClfromsampleswith9volumesofethanol(seeproteinprecipitationprotocol):resuspendedInclusionBodies.

7.Youcantryfirst8MofUrea,andifproteinissolubletiterdowninthenextexperimentstheureaconcentrationtillminimalureaisrequiredforproteinsolubilization.