Sphingosine Kinase 1
Sphingosine Kinase 1 (diluted in 100mM Tris pH7.5, 300mM NaCl, 10mM MgCl2, 2mM EGTA) is assayed in total volume of 20ul containing 100mM Tris ph 7.5, 400mM KCl, 10mM MgCl2, 2mM EGTA, 0.01mM ATP and 0.02mM Sphingosine. The enzyme is assayed for 40 min after which 20ul of ADP-Glo reagent is added. After a further incubation of 40 min, 40ul of Kinase Detection Buffer is added. The assays are incubated for 40 min and then read on PerkinElmer Envision for 1sec/well.
SPHK1_5.pdf (82.92 KB)