Contributor:SupryaJayadevDate:May25,1993Reagents:Lysisbuffer25mMTris-HCl,pH7.45mMEDTA1mMATP20µg/mlCLAP1mMPMSFBufferA10mMMgCl20.2MTris-HCl,pH7.40.2%TritonX-100BufferB0.2Msodiumacetate,pH5.00.2%TritonX-100BufferC0.2MTris-HCl,pH7.40.2%TritonX-100
Isolatingcellularenzyme1)Grow5x108cellsunderregulargrowthconditions.2)PelletcellsandwashonetimewithicecoldPBS.3)ResUSPendpelletin10mloficecoldlysisbuffer.4)Bombcells:20minutes@350psi,4°C5)Spinlysedcellmixtureat2,100rpm,4°Cfor10minute.6)Discardpelletandsetaside3mlofsupernatant.-->Supernatantfromthisstep="homogenate"7)Spinremainingsupernatant("7ml)@200,000xg,4°Cfor30minutes.centrifuge____________________rotor____________________rpm____________________time@plateau____________________8)Recoverbothsupernatantandpelletseparately.-->Supernatant="cytosol"9)Resuspendpelletin3mllysisbuffer.-->Thisfraction="membrane"
Preparingsubstrate10)Resuspenddried14C-SMinappropriatebuffertoget20nmoles,2x105cpm,100µlpersample.BufferA:Forneutral,Mg-dependentenzyme.BufferB:Foracidicenzyme.BufferC:Forneutral,Mg-independentenzyme.11)Vortexvigorouslyandsonicateifneccessary.-->Makesurelipidisfullysolubilized!!Assayingenzymeactivity12)Mixcellularenzymewithinducergently.-->Thevolumeofthismixshouldbe100µl.13)Pre-incubatefor10minutesat37°C.14)Add100µl14C-SMmixtoeachsampleandmixgently.15)Incubatereactionfor15minutes.16)Stopreactionsbyadding1.5mlofchloroform:methanol,1:2.17)Vortex,add0.2mlofwaterandvortex.18)Add0.5mlchloroformandvortextobreakphases.19)Add0.5mlwaterandvortex.20)Spinsamplesat3,000rpmfor5minutes.21)Count950µloftheupperphaseand0.5mlofthelowerphase.-->Thetotalvolumeofupper,aqueousphaseshouldequal1.9mlandthetotalvolumeofthelower,chloroformphaseshouldequal1ml.
