Ecl hrp substrate kit

Cells were lysed with ice-cold PRO-PREP™ protein extract solution (iNtRON, Sungnam, Gyunggi, Korea) and the protein concentration was determined by using the bicinchoninic acid (BCA) procedure (Thermo Scientific, Sunnyvale, CA, USA). Equal amounts of protein were separated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk in 100 mM Tris-hydrochloride (HCl, pH 7.5), 150 mM sodium chloride (NaCl), and 0.2% Tween-20 (TBST) for 1 h at room temperature. The membranes were incubated with TBST containing 5% milk and the primary antibodies against anti-inducible nitric oxide synthase (iNOS), anti-cyclooxygenase (COX)-2, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with TBST, the blot was incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. The antigen was detected by using a Western Bright enhanced chemiluminescence (ECL) HRP substrate kit (Advansta, Menlo Park, CA, USA).

For detection of Pat1 in bacterial cell lysates, 30% preparation bacteria (preparation described above) were boiled in 1x SDS loading buffer (150 mM Tris pH 6.8, 6% SDS, 0.3% bromophenol blue, 30% glycerol, 15% β-mercaptoethanol) for 10 min, resolved on a 10% SDS-PAGE gel, then transferred to a PVDF membrane (Millipore, IPFL00010). The membrane was blocked overnight at 4 °C in TBS-T (20 mM Tris, 150 mM NaCl, pH 8.0, 0.1% Tween 20 (Sigma, P9416) plus 5% dry milk (Apex, 20–241). Affinity-purified anti-Pat1 antibody was diluted 1:1000 in TBS-T plus 5% dry milk and incubated with the membrane overnight at 4 °C. Anti-RickA^{101 (link)} was used as a loading control by diluting serum 1:2,000 in TBS-T plus 5% dry milk and incubating at room temperature for 1 h. Membranes were washed with TBS-T for 5 × 5 min intervals at room temperature. Secondary antibody goat anti-rabbit HRP (Santa Cruz Biotechnology, sc-2004) was diluted 1:3,000 in TBS-T plus 5% dry milk and incubated at RT for 30 min, followed by washes with TBS-T. To detect secondary antibodies, ECL HRP substrate kit (Advansta, K-12045) was added to the membrane for 45 s at room temperature and developed using Biomac Light film (Carestream, 178-8207).