ADP-ribosyl cyclase activity measurement

Frozen egg/embryo samples were thawed, homogenized in a buffer comprising 20 mM HEPES (pH 7.2) supplemented with complete™ EDTA free protease inhibitors and sonicated (5 x 5s) to form a 25% v/v homogenate. Homogenates were diluted 10-fold in to a reaction mix containing a final concentration of either 20 mM HEPES (pH 7.2) and 1 mM NAD (for cyclase activity measurements) or 50 mM nicotinic acid (pH 4.8) and 1 mM NADP (for base-exchange activity measurements). The reactions were allowed to proceed for up to 3h. To terminate the reactions, samples were diluted 10-fold with water and heated at 60°C for 5 min. Samples were then centrifuged for 3 min at 21000 x g to remove any particulate matter. The products in the supernatants were resolved on an AG MP1 anion exchange HPLC column using a concave-up gradient of trifluoroacetic acid as described in (Churamani et al., 2007). Protein concentration was measured using the bicinchoninic acid protein assay kit (Pierce) and bovine serum albumin as the standard according to manufacturer’s instructions.