In vitro Sphingomyelinase Assay

Reagents: Lysis buffer 25 mM Tris-HCl, pH 7.4 5 mM EDTA 1 mM ATP 20 µg/ml CLAP 1 mM PMSF Buffer A 10 mM MgCl2 0.2 M Tris-HCl, pH 7.4 0.2 % Triton X-100 Buffer B 0.2 M sodium acetate, pH 5.0 0.2 % Triton X-100 Buffer C 0.2 M Tris-HCl, pH 7.4 0.2 % Triton X-100

Isolating cellular enzyme 1) Grow 5 x 108 cells under regular growth conditions.2) Pellet cells and wash one time with ice cold PBS.3) Resuspend pellet in 10 ml of ice cold lysis buffer.4) Bomb cells: 20 minutes @ 350 psi, 4°C5) Spin lysed cell mixture at 2,100 rpm, 4°C for 10 minute.6) Discard pellet and set aside 3 ml of supernatant.--> Supernatant from this step = "homogenate"7) Spin remaining supernatant (" 7 ml) @ 200,000 xg,4 °C for 30 minutes.centrifuge ____________________rotor ____________________rpm ____________________time @ plateau ____________________8) Recover both supernatant and pellet separately.--> Supernatant = "cytosol"9) Resuspend pellet in 3 ml lysis buffer.--> This fraction = "membrane"Preparing substrate10) Resuspend dried 14C-SM in appropriate buffer to get 20 nmoles, 2 x 105 cpm, 100 µl per sample.Buffer A: For neutral, Mg-dependent enzyme.Buffer B: For acidic enzyme.Buffer C: For neutral, Mg-independent enzyme.11) Vortex vigorously and sonicate if neccessary.--> Make sure lipid is fully solubilized!!Assaying enzyme activity12) Mix cellular enzyme with inducer gently .--> The volume of this mix should be 100 µl.13) Pre-incubate for 10 minutes at 37 °C.14) Add 100 µl 14C-SM mix to each sample and mix gently .15) Incubate reaction for 15 minutes.16) Stop reactions by adding 1.5 ml of chloroform:methanol, 1:2.17) Vortex, add 0.2 ml of water and vortex.18) Add 0.5 ml chloroform and vortex to break phases.19) Add 0.5 ml water and vortex.20) Spin samples at 3,000 rpm for 5 minutes.21) Count 950 µl of the upper phase and 0.5 ml of the lower phase.--> The total volume of upper, aqueous phase should equal 1.9 ml and the total volume of the lower, chloroform phase should equal 1 ml.