Chemiluminescence blotting substrate

Total protein was extracted from transiently-expressing HEK-293T cells with cell lysis buffer (0.1 M Tris-HCl pH 7.8, 0.5% Triton X-100) containing cOmplete Mini EDTA-free protease inhibitor (Roche). Concentration of protein extracts was determined using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories) according to the manufacturers’ instructions. Protein samples (∼250 μg) were resolved by sodium dodecylsulphate-polyacrylamide gel electrophoresis (7.5% polyacrylamide) and transferred to a polyvinylidene fluoride membrane at 70 mA for 20 hours at 4°C. The membrane was blocked using 5% skim milk in TBST before addition of the primary antibodies; 34C (Sigma) for detection of RyR1 or anti-α-tubulin (Sigma) as a loading control, followed by horseradish peroxidase-conjugated anti-mouse secondary antibody (Jackson laboratories). Proteins were visualized using an X-ray developer after brief exposure with chemiluminescence blotting substrate (Roche) according to the manufacturer’s instructions. Successful expression of RyR1 protein was assessed by visual inspection of the film image, comparing the RyR1 band with the matching tubulin band.

Peptides were synthesized by Fmoc chemistry with the C-terminus linked to Whatman 50 cellulose membrane in a spot format (JPT Peptide Technologies) (Frank, 2002 (link)). The membrane was rinsed with methanol, washed with Tris-buffered saline (TBS), pH 8.0, and then incubated with 1% blocking solution (Roche) in TBS for two hours. Antibodies at the concentration of 2–8 µg/mL were incubated with the membrane for three hours before washing with TBS containing 0.05% Tween 20 and then 0.5% blocking solution in TBS. Bound antibodies were detected by using horse radish peroxidase (HRP)-conjugated rabbit anti-mouse Igs antibody (DAKO) and a chemiluminescence blotting substrate (Roche). Signals were detected by exposing the membrane to X-ray film.

Cells were collected and lysed with NP-40 lysis buffer (50 mM HEPES, pH 7.4, 200 mM KCl, 0.3% NP-40, 10% glycerol, 1 mM EGTA, 1 mM MgCl₂, 0.5 mM DTT, 0.5 μM microcystin, and 10 μg/ml concentrations of leupeptin, pepstatin, and chymostatin) on ice for 10 min and debris was removed by centrifugation. Protein concentrations in total cell lysates were measured using a Bradford protein assay kit before boiling in Laemmli buffer. Equal volumes of protein samples were loaded, separated by a 12% SDS-PAGE gel, and transferred to a PVDF membranes. The membranes were incubated overnight at 4°C with the primary antibodies, followed by incubation with the appropriate secondary antibodies. HRP-conjugated anti-mouse IgG or anti-rabbit IgG were used as secondary antibodies. Protein bands were detected using chemiluminescence blotting substrate (Roche, Switzerland) and X-ray film.