Goat anti rabbit igg antibody conjugated to horseradish peroxidase

Flash frozen hearts were homogenized at 4 °C in radio-immunoprecipitation assay (RIPA) buffer (in mmol·L⁻¹: Tris 10, NaCl 140, EDTA 5, phenylmethanesulfonylfluoride PMSF 1 with Triton X-100 1%, deoxycholate 1%, sodium dodecyl sulfate (SDS) 0.1%, and in μg/mL: aprotinin 10, leupeptin 10, pepstatin 10, at pH 7.4) using a glass dounce tissue grinder. A mitochondria isolation kit for tissue (Abcam, Paris, France) was used to prepare enriched mitochondrial fractions, which were stored at −80 °C until use. Proteins (25 μg) were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto a nitrocellulose membrane and incubated with the following antibodies: rabbit polyclonal anti VDAC-1 antibody (1/1000; Abcam, Paris, France), rabbit polyclonal anti-phospho-Ser-58 CcOX IV-1 antibodies (1/500; Phospho Solutions, Aurora, CO, USA). Blots were developed with enhanced chemiluminescence (ECL) Plus reagent with goat anti-rabbit IgG antibody conjugated to horseradish peroxidase for chemiluminescent detection (Cell Signaling Technology, Beverly, MA, USA).

Fresh cells were lysed on ice for 30 minutes using lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1 mM Ethylenediaminetetraacetic acid disodium salt (EDTA). The protein concentration was examined using the bicinchonininc acid (BCA) protein assay kit (Thermol). Proteins were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride membranes (Millipore). The membrane was blocked in the blocking buffer included 0.1 M Tris-HCL, 0.9% NaCl, 0.8% KCl, 0.5% Tween-20 (TBST) and 5% nonfat silk milk and incubated with antibodies against human LEF1 (Cell Signaling Technology), β-actin (Cell Signaling Technology) at a 1:1000 dilution and CYLD (Cell Signaling Technology) at a 1:250 dilution, followed by incubation with goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (Cell Signaling Technology) at a dilution of 1:5000. Enhanced chemiluminescence was used to detect proteins.

The WT strain containing pASK-H-NS-3FLAG plasmid was grown in LPM for 20 h in the presence or absence of anhydrotetracycline (AHT) for 4 h. The cells were washed and ~5 × 10⁷ colony-forming units were pelleted and re-suspended in Laemmli sample buffer, boiled for 5 min, and then separated on SDS-PAGE. Proteins on the gel were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking in Tris-buffered saline (TBS) plus 5% non-fat dry milk for 1 h, membranes were probed with anti-FLAG monoclonal antibody (Sigma). Membranes were washed and probed with secondary antibody (anti-mouse IgG conjugated with peroxidase) (Sigma). The immune complexes were detected via chemiluminescence using Western Lightning™ (PerkinElmer), and then exposed to XAR Biofilm (Kodak). For immunoblot on H-NS, WT and ΔrpoE strains were grown in LB to log phase, or in LPM for 4 h or 20 h. Affinity purified rabbit anti-H-NS antibody was used as primary antibody, and goat anti-rabbit (IgG) antibody conjugated to horseradish peroxidase (Cell Signaling Technology) was used as the secondary antibody. In both cases, DnaK was probed at the same time as loading control.