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https://doi.org/10.1016/s0076-6879(97)80120-6
Copy DOIJournal: Methods in Enzymology | Publication Date: Jan 1, 1997 |
Citations: 21 |
Publisher Summary This chapter describes a bioassay for cyclic ADP-ribose (ADPR) on the basis of its Ca 2+ -releasing activity in homogenates prepared from sea urchin eggs. Substances that could potentially interfere with the Ca 2+ release bioassay, such as Ca 2+ , inositol 1,4,5-triphosphate, and NAD + , must be removed from the sample. Cyclic ADP-ribose in the extracts is purified by a two-step system consisting of phenylboronate chromatography followed by anion-exchange chromatography on AG MP-1. Strongylocentrotus purpuratus eggs are obtained by stimulating ovulation of a female sea urchin with a 1-ml injection of 0.5 M KCI and washed once in artificial seawater, twice in Ca 2+ -free seawater containing 1 m M ethylene glycol tetraacetic acid (EGTA), twice in Ca 2+ -free seawater without EGTA, once with the homogenization buffer, and resuspended with the same medium to 25%. The sensitivity of the bioassay can be increased by pretreating the homogenate with caffeine, which is known to potentiate cADPR-induced Ca 2+ release.
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