| Assay Method Information | |
| | In Vitro cPLA2 Assay |
| Description: | Assay for cPLA2 activity was performed by the use of sonicated vesicles of 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphorylcholine (100 μM) containing 100,000 cpm of 1-palmitoyl-2-[1 14C]arachidonoylsn-glycerol-3-phosphorylcholine in 100 mM Hepes, pH 7.5, 80 μM Ca2, 2 mM dithiothreitol, and 0.1 mg/ml BSA as described. Following a 35-mM incubation at 37° C., the reaction was terminated (derived from Wijkander et al). The lower phase was separated by thin layer chromatography, and the spot corresponding to free [1-14C]arachidonic acid was visualized by digital imaging and quantified with a PhosphorImager (Fuji Instruments). The source of cPLA2 enzyme was recombinant overexpression of the human gene for group IVa PLA2 in baculovirus insect cell expression system, as described in Abdullah et al. Wijkander, J., and Sundler, R. (1991) Eur. J. Biochem. 202, 873-880 Abdullah, K., et al. (1995) Human cytosolic phospholipase A2 expressed in insect cells is extensively phosphorylated on Ser-505. Biochim Biophys Acta. 1995 May 11; 1244(1):157-64. |
| Affinity data for this assay | |
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