Rapid cbb kanto

Protein concentration was determined with a Quick Start Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The apparent molecular mass of the enzymes was determined by SDS-PAGE in a 10–15 % polyacrylamide gel gradient as described by Laemmli. ¹⁷⁾ A BenchMark Ladder (Invitrogen, Carlsbad, CA, USA) provided the standard protein markers. The molecular mass of the native enzyme was determined by native-PAGE in an 8–25 % polyacrylamide gel gradient as described by Davis. ¹⁸⁾ The HMW marker kit (GE Healthcare, Chicago, IL, USA) provided the standard protein markers. The p I for thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and albumin (66 kDa) in the protein marker mix were 4.5, 4.5, 5.4, 5.0, and 4.9, respectively. The apparent p I of the enzyme was determined by isoelectric focusing on a Phastgel IEF 3–9 using the Phastsystem (GE Healthcare, Chicago, IL, USA). The proteins were stained with Rapid CBB KANTO (Kanto Chemical Co. Inc., Chuo-ku, Japan).

Protein concentration was determined using Quick Start Bradford Protein Assay (Bio-Rad, USA). The apparent molecular mass of the enzymes was determined using SDS-PAGE in 10–15 % gradient polyacrylamide gels as described by Laemmli.¹¹⁾ (link) Benchmark Ladder (Invitrogen) was used as standard protein markers. The molecular mass of the native enzymes was determined using native-PAGE in 8–25 % gradient polyacrylamide gels as described by Davis.¹²⁾ HMW Marker Kit (GE Healthcare) was used as standard protein markers. The pI values of thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and albumin (66 kDa) present in the protein marker are 4.5, 4.5, 5.4, 5.0, and 4.9, respectively. The apparent pI of the enzymes was determined using isoelectric focusing on a Phastgel IEF 3–9 and the Phastsystem (GE Healthcare). Proteins were stained using Rapid CBB KANTO (Kanto Chemical, Japan).

Mouse tissues or cultured cells were lysed using lysis buffer (125 mmol/l NaCl, 20 mmol/l Tris–HCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate; pH 7.4) followed by centrifugation at 15,000 rpm for 5 min at 4 °C to remove undissolved debris. The lysates were then sonicated (Handy Sonic UR-20 P; Tomy Seiko Co, Ltd) on ice. Protein concentrations of the lysates were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were loaded onto SDS–PAGE gels, transferred to polyvinylidene difluoride membranes (Wako), and incubated overnight with the primary antibody of interest. Alkaline phosphatase–conjugated secondary antibody (Cell Signaling) and 5-bromo-4-chloro-3-indolyl-phosphate–nitro blue tetrazolium (Wako) were used to detect the target proteins. The primary antibodies used for immunoblotting are listed in Table S2. Coomassie brilliant blue staining was conducted using Rapid CBB KANTO (KANTO Chemical Co) according to the manufacturer’s instructions.