Assay Report Card

ID: ALA3706130

Type: Binding

Description: Inhibition Assay: To prepare the substrate, 25-50 uCi of [3H]trioleoylglycerol (in toluene), 6.8 umol of unlabeled trioleoylglycerol and 0.6 mg of phospholipids (phosphatidylcholine/phosphatidylinositol 3:1 w/v) are mixed, dried with N2 and then taken up in 2 ml of 0.1 M KPi (pH 7.0) by ultrasound treatment (Branson 250, microtip, setting 1-2, 2x1 min with an interval of 1 min). After addition of 1 ml of KPi and renewed ultrasound treatment (4x30 sec on ice with intervals of 30 sec), 1 ml of 20% BSA (in KPi) is added (final concentration of trioleoylglycerol 1.7 mM). For the reaction, 100 ul of substrate solution are pipetted into 100 ul of HSL solution (HSL prepared as above, diluted in 20 mM KPi, pH 7.0, 1 mM EDTA, 1 mM DTT, 0.02% BSA, 20 ug/ml pepstatin, 10 ug/ml leupeptin) and incubated at 37 C. for 30 min. Addition of 3.25 ml of methanol/chloroform/heptane (10:9:7) and of 1.05 ml of 0.1 M K2CO3, 0.1 M boric acid (pH 10.5) is followed by thorough mixing.