Peroxidase conjugated goat anti rabbit igg h l

The protein was lysed in lysis solution supplemented with phosphatase inhibitor, protease inhibitor, and phenylmethylsulfonyl fluoride, followed by the measurement of protein concentration using a BCA kit (Thermo Fisher Scientific). Then, 10–20 µg protein was sampled on 8–12% 30% acrylamide-Bis gel and then transferred onto 0.22 µm PVDF membranes. Subsequently, the membranes were treated with 5% skim milk for 1 h and incubated with antibodies GAPDH (ab8245, Abcam), SP1 (ab227383, Abcam), GPX4 (ab125066, Abcam), and SLC7A11 (ab175186, Abcam) overnight. All antibodies were diluted according to the instructions. Next day, the membranes were cultured with the secondary antibody peroxidase-conjugated goat anti-rabbit IgG (H&L) (#111035003, Jackson ImmunoResearch, USA) for 1 h and developed with luminescent liquid (Thermo Fisher Scientific). Image J was used for analysis. The relative protein content was expressed as the gray value of the corresponding protein band/the gray value of GAPDH protein band. The experiment was repeated three times.

Both rabbit and horse polyclonal antibodies raised against the venom of N. atra were donated by the Centers for Disease Control, Taiwan. These antivenoms were further affinity-purified by LTK BioLaboratories Co., Ltd. with an antigen-immobilized column system and dialyzed with PBS. Peroxidase-conjugated goat anti-rabbit IgG (H + L) and goat anti-horse IgG (H + L) were purchased from Jackson ImmunoResearch Inc. (West Grove, PA, USA). SureBlue Reserve™ TMB Microwell Peroxidase Substrate was purchased from SeraCare Life Sciences Inc. (Milford, MA, USA). Bolt™ 4–12% Bis-Tris Plus Gels and Novex^® Sharp Pre-stained Protein Standard were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Immobilon-PSQ PVDF membrane and the chemiluminescent HRP substrate used in Western blotting were obtained from Merck Millipore (Burlington, MA, USA). Flat-bottom microtitration plates were purchased from Corning Inc. (Corning, NY, USA). All other chemicals were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Proteins from cell lysates were separated by 10% acrylamide SDS–PAGE and subjected to western blot analysis using polyclonal rabbit anti-NP antibody (catalogue number: PA5-32242; Thermo Fisher; diluted 1:2,500), monoclonal mouse anti-β-tubulin antibody (catalogue number: T4026; Sigma-Aldrich; diluted 1:1,000) and the corresponding secondary antibodies IRDye 680RD goat anti-rabbit IgG (H+L) (LI-COR Biosciences; diluted 1:5,000) or IRDye 800CW goat (polyclonal) anti-mouse (LI-COR Biosciences; diluted 1:5,000). Signals were visualized by Odyssey Imaging System (LI-COR Biosciences). Proteins of purified viral particles were separated as described above and detected by western blot analysis using polyclonal rabbit anti-NP, polyclonal mouse anti-M1 (provided by Dr. Jovan Pavlovic; diluted 1:100) and corresponding peroxidase-conjugated goat anti-mouse IgG (H+L) (Jackson ImmunoResearch; diluted 1:5,000) or peroxidase-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch; diluted 1:5,000) antibodies. Signals were detected with an Odyssey Imaging System (LI-COR Biosciences). Uncropped western blots can be found in Supplementary Fig. 9.