Shrimp ribosomal protein purification

Description

To better understand the differential expression and post-translation of modification of host proteins after WSSV infection, we established a ribosomal protein purification platform. This technology is used to support ribosomal protein analysis.

Brief protocol

Ribosomal proteins were prepared as described previously (Siegmann and Thomas 1987; Williams, Werner-Fraczek et al. 2003; Chang, Szick-Miranda et al. 2005) with the following modification. Frozen shrimp stomachs were ground to a fine powder under liquid nitrogen with a mortar and pestle. The powder was then homogenized in ribosome extraction buffer（200 mM Tris-HCl, pH 7.5, 200 mM KCl, 25 mM EGTA, 36 mM MgCl2, 1 mM sodium molybdate, 1 mM dithiothreitol﹝DTT, in 10 mM sodium acetate, pH 5.2﹞, 50 mg/ml chcloheximide, 50 mg/ml chloramphenicol, 1 %﹝v/v﹞Triton X-100, 1%﹝v/v﹞ Brij 35, 1%﹝v/v﹞Tween-40, and 1%﹝v/v﹞NP-40）and incubated on ice for 20 min. After centrifugation at 15000 rpm for 10 min at 4℃, the supernatant was layered onto 2 ml 1 M sucrose cushion made in 400 mM Tris-HCl, pH 7.5, 200 mM KCl, 5 mM EGTA , 36 mM MgCl2, 1.3 M Sucrose, 1mM sodium molybdate, 1 mM dithiothreitol﹝DTT, in 10 mM sodium acetate, pH 5.2﹞, 50 mg/ml chcloheximide, and 50mg/ml chloramphenicol. Ribosomes were obtained by centrifugation at 4℃ in a Hitachi RPS56T rotor at 149000 g for 18 hr. The ribosomes pellet were resuspended in Staehlin A buffer（20 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2 , 1 mM sodium molybdate, 1 mM dithiothreitol﹝DTT, in 10 mM sodium acetate, pH 5.2﹞）for 1 hr at 4℃ and quantified by measuring A260. In order to remove the rRNA from ribosomes, the ribosomes sample were vortexed for 1 hr at 4℃ by addation of 0.1 volumes of 1 M MgCl2 and 2 volumes of glacial acetic acid. After centrifugation at 15000 rpm for 10 min at 4℃, the supernatant, which contained the ribosomal proteins, was collected and precipitated with 5 volumes of acetone at -80℃ for 24 hr. The sample was spun at 15000 rpm for 10 min at 4℃ and washed several times with acetone, and then dried in air and froze at -80℃.