Goat anti rabbit igg hrp conjugate

Immunoblotting was performed using 4–12% gradient gels through MOPS SDS-PAGE, as previously described [54 (link), 55 (link), 57 (link)–60 (link)]. Normalization of protein content was assessed using the Bradford Method [56 (link)]. Primary antibodies utilized in the study included the following: total OXPHOS Blue Native WV Antibody Cocktail (ab110412) (anti-NDUFA9 (complex I, ab14713, Abcam, Cambridge, MA), anti-SDHA (complex II, ab14715, Abcam), anti-UQCRC2 (complex III, ab14745, Abcam), anti-COX IV (complex IV, ab14744, Abcam), and anti-ATP5A (complex V/ATP Synthase, ab14748, Abcam)), anti-ATP5F1 (complex V/ATP Synthase, ab117991, Abcam), and anti-VDAC (#4866, Cell Signaling Technology, Danvers, MA). Goat anti-mouse IgG (H&L) horseradish peroxidase (HRP) conjugate 1:10,000 (Thermofisher Scientific) and goat anti-rabbit IgG HRP conjugate 1:5000 (Abcam) were used as the secondary antibodies. Normalization of protein content was through VDAC expression. Chemiluminescence quantified with Radiance Chemiluminescent Substrate (Azure Biosystems, Dublin, CA), per manufacturer’s instructions and imaged using the G:Box Bioimaging system (Syngene, Frederick, MD). GeneSnap/GeneTools software (Syngene) was used to acquire images. Densitometry was analyzed using Fiji Software (NIH, Bethesda, MD).

A 15% acrylamide gel was prepared, and a sample buffer was added to each of 23 fractions in which cytochrome oxidase activity was observed and electrophoresed at 30 mA. Blue Star Prestained Protein Marker (NIPPON Genetics) was used as a marker. Proteins in the gel were transferred to nitrocellulose filters (Bio-Rad) by applying electricity at 20 V for 16 h in Tris-glycine-methanol buffer [25 mM Tris, 192 mM glycine, and 20% (v/v) methanol (pH 8.3)] using a Mini Trans-Blot^® Cell manufactured (Bio-Rad). Western blots were performed as described previously (Morino et al., 2008 (link)). The β subunit-His₆ protein of ATP synthase was detected by anti-His antibody HRP conjugate (Qiagen). For detection of the CtaC protein, rabbit anti-CtaC polyclonal antibody (Eurofins Genomics) was used as a primary antibody and goat anti-rabbit IgG-HRP conjugate (Abcam) was used as a secondary antibody. ECL solution (Promega) was the usual detection reagent. A quantitative imaging system, ChemiDoc^TM XRS⁺ (Bio-Rad) and a PC application software, Quantity One were used for the detection and analysis of chemiluminescence images.

Anti-human IL-17R rabbit polyclonal antibody (H-168 clone) was purchased from Santa Cruz Biotechnology Inc., Dallas, TX, USA. Goat anti-rabbit IgG-HRP conjugate was obtained from Abcam plc., Cambridge, UK. The monoclonal anti-poly-histidine-HRP antibody produced in mouse was obtained from Sigma-Aldrich, St. Louis, MO, USA. Rabbit polyclonal to human IL17A was obtained from Abcam plc., Cambridge, UK. Cy5-conjugated goat anti-rabbit IgG F(ab´)₂ fragment was obtained from Jackson ImmunoResearch Laboratories, West Grove, PA, USA.
Recombinant human IL-17 RA/IL-17 R Fc Chimera was obtained from R&D Systems, Minneapolis, MN, USA. Human IL-17A was purchased from Cell Signaling Technology, Danvers, MA, USA. Streptavidin-phycoerythrin was purchased from eBioscience, San Diego, CA, USA. Streptavidin-HRP conjugate was obtained from Thermo Scientific, Rockford, IL, USA.