PROTEIN EXTRACTION

Total protein from coelomocytes was extracted and purified for subsequent SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Total coelomic fluid (12.5-25 ml depending on the size of the sea urchin) was collected after removal of Aristotle’s Lantern and coelomocytes were pelleted at 3000 x g for 5 min at 4oC. The cell pellet was lysed in lysis buffer (5 ml; 7 M urea, 2 M thiourea, 1% 3-[4-heptyl]phenyl-3-hydroxypropyldimethylammoniopropanesulfo-nate [ASB-C7BzO], 40 mM Tris base, 15 mM acrylamide [Bio-Rad], 10 mM tributyl phosphine [TBP], and 1x complete protease inhibitors [Roche]) using a French press (Thermo Spectronic). The lysate was incubated for 1 h at room temperature to allow complete reduction and alkylation of cysteines. Cell debris was pelleted by centrifugation for 10 min at 5000 x g, the supernatant was transferred to a fresh tube and proteins were precipitated for 30 min at room temperature with five volumes of acetone and centrifuged for 10 min at 5000 x g. The acetone supernatant was decanted, the pellet was air dried for 5 min and dissolved in 0.5-1.5 ml of sample buffer (7 M urea, 2 M thiourea, 1% C7BzO). Finally, the protein solutions were desalted using Micro Biospin tubes (Bio-Rad) and the eluates stored at -20 C.

Sham Nair 2014