Microbca protein assay kit 23235

Spinal cord tissues were weighted and homogenized in 2 weight-volume (mg/μl) of PBS by sonication (Output 2, 10–20 times, BRANSON Sonifier 450). One-hundred microliters of homogenates and 700 μl of Lysis buffer A’ [25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1% Triton X-100, Complete Protease Inhibitor Cocktail (Roche), Phosphatase inhibiter Cocktail I (SIGMA)] were mixed, followed by centrifugation at 23,000×g for 20 min at 4 °C. Supernatant was collected as 1% Triton X-100 soluble fraction. The insoluble pellet was washed by mixing with 600 μl of Lysis buffer A’ and centrifuged again, and resulting pellet was suspended in 400 μl of Lysis buffer B′ [25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5% sodium dodecyl sulfate (SDS)], sonicated (Output 2, 5–10 times), and used as 1% Triton X-100 insoluble fraction. Protein concentration was determined by Micro BCA (Thermo, MicroBCA Protein Assay kit #23235). Each protein fraction was diluted by the corresponding lysis buffer and adjusted its concentration at 0.75 μg/μl. One-hundred microliters of diluted samples were mixed with 50 μl of 3xSDS buffer [187 mM Tris-HCl (pH 6.8), 6% SDS, 30% glycerol, 0.015% bromophenol blue (BPB), 15% 2-melcaptoethanol] and heated for 5 min at 95 °C.