反应详情

Soluble Shh was labeled with 3H-palmitic acid in a cell-free system using a modified version of a published procedure (24). A crude microsomal fraction from rat liver was prepared by subjecting a liver homogenate to sequential centrifugation at 3000×g for 10 min, 9000×g for 20 min, and 100,000×g for 30 min. The 100,000×g pellet was suspended in 10 mM HEPES pH 7.4, 10% sucrose and again centrifuged at 100,000×g for 20 min. The final pellet (derived from 10 g of liver) was suspended in 3 mL of 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 μg/mL leupeptin, 0.15% Triton X-100, aliquoted, and stored at −70° C. Reactions containing 3 μg Shh, 1 μL of rat microsomes, 50 ng/mL Coenzyme A (Sigma), 0.3 mM ATP, 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 μg/mL leupeptin, and 0.5 μCi-[9,10-3H]-palmitic acid (50 Ci/mmol; New England Nuclear) were performed at room temperature for 1 h. Reactions were stopped with reducing electrophoresis sample buffer, subjected to SDS-PAGE on a 10-20% gradient gel, and visualized by fluorography.

1. customA crude microsomal fraction from rat liver was prepared
2. wait9000×g for 20 min
3. wait100,000×g for 30 min
4. waitagain centrifuged at 100,000×g for 20 min
5. customstored at −70° C
6. waitwere performed at room temperature for 1 h