Horseradish peroxidase conjugated goat anti mouse igg

Total protein was extracted from the leaves of 4‐week‐old wild‐type R108 and transgenic 35S:MtING2‐3xFLAG Medicago plants grown in VLD conditions using the trichloroacetic acid/acetone method as described previously (Isaacson et al., 2006 (link)). Protein was separated by SDS‐PAGE (20 μg per lane) and transferred onto 0.2‐μm nitrocellulose membrane (Whatman, UK). The membrane was blocked in Tris‐buffered saline + 0.1% Tween 20 with 5% low‐fat milk powder. MtING2‐3xFLAG protein was detected using 1 μg/μl of mouse monoclonal anti‐FLAG (Sigma, F1804) and 80 pg/μl of horseradish peroxidase‐conjugated goat anti‐mouse IgG (Jackson ImmunoResearch Laboratories, USA). Images were captured using an Amersham Imager 600 (General Electric, USA) with Pierce SuperSignal West Femto Substrate (Thermo Fisher, USA).

Western blot analysis was performed on the proteins extracted from the total lysate, or from the nuclear, cytosolic, or mitochondrial fractions of hippocampal CA3 tissues. The primary antisera was used, including a mouse monoclonal or polyclonal antiserum, against the SIRT1 (sc-74465, Santa Cruz Biotechnology, Dallas, TX, USA), Tfam (ab131607, Abcam, Cambridge, UK), or COX1 (ab14705, Abcam); or a rabbit polyclonal antiserum against PGC-1α (sc-13067, Santa Cruz Biotechnology), NRF1 (sc-33771, Santa Cruz Biotechnology), COX IV (ab16056, Abcam), or β-actin (ab8227, Abcam). This was followed by incubation with the secondary antisera, including horseradish peroxidase-conjugated goat anti-mouse IgG (115-035-003, Jackson ImmunoResearch, West Grove, PA, USA) for SIRT1, Tfam, and COX1; or goat anti-rabbit IgG (111-035-045, Jackson ImmunoResearch) for PGC-1α, NRF1, COX IV, and β-actin. The specific antibody–antigen complex was detected by an enhanced chemiluminescence western HRP substrate (Merck Millipore, Billerica, MA, USA). The amount of protein was quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA) and was expressed as the ratio relative to β-actin protein or COX IV (as the mitochondrial loading control).

Overnight MOPSg cultures of BL21(DE3) containing plasmids expressing His₆-tagged RcrR wild-type, His₆-tagged RcrR-4C-S, or the empty vector control were diluted into fresh MOPSg and incubated at 37°C and 300 rpm until mid-log phase. When the cultures reached at A_{600 nm} ~0.35, protein expression was induced by adding 100 μM IPTG. When cells reached A_{600 nm} ~0.55, cultures were either left untreated or treated with 2.5 mM HOCl for 15 min. Cells equivalent to 8 mL of A_{600 nm} = 1 were harvested by centrifugation and incubated in 75 μL of lysis buffer (10 mM KPi [pH 6.5], 1 mM EDTA, 20% [wt/vol] sucrose, 2 mg/mL lysozyme, 50 U/mL benzonase) supplemented with 0.8 M iodoactamide for 30 min. After a quick freeze-thaw cycle and the addition of 360 μL buffer A (10 mM KPi [pH 6.5], 1 mM EDTA), cells disrupted using 0.5 mm glass beads (BioSpec Products) for 30 min at 8°C. The cell lysates were collected, proteins separated by 12% nonreducing SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific). The membrane was blocked with Tween20-containing tris-buffered saline buffer containing 3% milk powder and 1% bovine serum albumin, incubated overnight with anti-His antibody (Cell Biolabs) and finally with horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratory) for 1 h.

The cytosol proteins of cancer tissues and adjacent non-tumor lung tissues were mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample lysis buffer (2% SDS; 20% glycerol; 50mM Tris-HCl, pH 6.8). Thirty micrograms of protein from each matched tumor and non-tumor sample was resolved in continuous 12% Tris-HCl SDS-PAGE gel. Proteins were transferred to a Polyvinylidene Fluoride (PVDF) Membrane (0.45 μm; Bio-Rad, Hercules, CA, USA) and was blocked for 15 min at 25°C in TBS-T (20 mM Tris-HCl, pH 7.5; 50 mM NaCl; 0.05% Tween-20) buffer containing 5% skimmed milk. The PVDF membrane was incubated for 120 minutes at room temperature with a 1:2000 dilution antibody against CCT5 (Novus Biologicals, CO, USA). As secondary antibody, horseradish peroxidase–conjugated goat anti–mouse IgG (Jackson, PA, USA) was applied at a 1:5,000 dilution. Immunoreactive bands were detected with an ECL kit (YuanPingHao Bio, Beijing, China) according to the manufacturer's instructions and followed by autoradiography. Western blot films were scanned in transmittance mode in a ScanMaker S430 scanner using 300 dpi.

Lumbar 4–5 spinal dorsal horns were collected and protein was extracted. 50 μg of protein from each group was then loaded and separated by SDS-PADGE, transferred to a PVDF membrane, blocked with 5% skim milk in TBST, and incubated overnight with primary antibodies at 4 °C. Primary antibodies include, rabbit anti-Hv1 (1:2000; AHC-001, RRID:AB_10917155, Alomone labs) and GAPDH (1:1000; sc-32233, RRID:AB_627679, Santa Cruz Biotechnology). Membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000; 111-036-045, RRID:AB_2337943, Jackson ImmunoResearch Labs) and horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000; 115-035-003, RRID:AB_10015289, Jackson ImmunoResearch Labs) for 1 h at room temperature. Membranes were then treated with West Pico substrate (34078, Thermo Fisher Scientific) and chemiluminescence signal was detected with a G:BOX Chemi XRQ gel doc (Syngene, Frederick, MD). Optical density of each band was then determined using Fiji, (NIH).