Horseradish peroxidase hrp conjugated secondary antibody

Levels of IgG, IgA, and IgM antibodies in human sera and animal sera were measured by an indirect ELISA with recombinant proteins (S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2 and S, N, E, M, and 3CLpro of SARS-CoV), and whole inactivated virus antigens (SARS-CoV-2, HCoV NL63, CCoV 1-71, and H1N1 influenza virus [A/California/2009/07]), which were prepared by density-gradient purification, followed by γ-irradiation or β-propiolactone inactivation. Heat-inactivated sera (56 °C for 30 min) were added to the wells of antigen-coated plates and then detected by the respective horseradish peroxidase (HRP)-conjugated secondary antibodies (SouthernBiotech, Birmingham, AL, USA). TMB substrate (MilliporeSigma, Burlington, MA, USA) was added to produce a color change. The reaction was stopped with sulfuric acid, and optical density (OD) at 450 nm was read using the Synergy plate reader (Biotek, Broadview, IL, USA). The cutoff values of tests were defined as 3 times the average of the negative control samples for human sera, 3 times the average of the SPF samples for canine and feline sera, and 3 times the average of the preimmune murine sera.

LV tissue was homogenized in ice-cold lysis buffer containing PBS (pH 7.4), 0.5% Triton X-100, 300 mM NaCl, and protease/phosphatase inhibitor cocktail (Thermo Fisher) using the Next Advance Bullet Blender. Lysates were clarified at 16000×g for 5 min prior to protein concentration determination via BCA Protein Assay Kit (Pierce). Proteins were resolved by SDS/PAGE, transferred to nitrocellulose membranes (Bio-Rad), and membranes were blocked with 4% milk. Membranes were probed overnight with indicated primary antibodies for mouse monoclonal acetyl-lysine (Cell Signaling Technology; 9681), rabbit polyclonal acetyl-lysine (Cell Signaling Technology; 9441), acetyl-α-tubulin (Santa Cruz Biotechnology; sc-23950), α-tubulin (Santa Cruz Biotechnology; sc-23948), or total histone H3 (Cell Signaling Technology; 4499). Horseradish peroxidase (HRP)–conjugated secondary antibodies (Southern Biotech) were used at a concentration of 1:2000. SuperSignal West Pico chemiluminescence system (Thermo Scientific) and a ChemiDoc XRS+ imager (Bio-Rad) were used to detect protein expression.

Membranes were probed with primary antibodies for phosphorylated ERK (Cell Signaling Technology; 4370), total ERK (Santa Cruz Biotechnology; sc-153 or Cell Signaling Technology; 4696), sheep polyclonal anti-DUSP5 (13 (link)), rabbit anti-DUSP5 (Abcam; ab200708), c-Myc (Santa Cruz Biotechnology; sc-40), β-tubulin (Sigma; T8328), or α-tubulin (Santa Cruz Biotechnology; sc-23948 HRP). Horseradish peroxidase (HRP)-conjugated secondary antibodies (Southern Biotech) were used at a concentration of 1:2,000. SuperSignal West Pico chemiluminescence system (Thermo Scientific) and a FluorChem HD2 Imager (Alpha Innotech) were used to detect proteins. For indirect immunofluorescence, anti-DUSP5 antibody (Abcam) was diluted 1:100 in phosphate-buffered saline with 0.1% Tween-20 (PBS-T) containing 1% BSA and incubated overnight. Secondary goat anti-rabbit IgG conjugated to Alexa Fluor 594 (Thermo Fisher; A-11012) and DAPI were used for detection of DUSP5 and nuclei, respectively.

Parasites were maintained in mice as previously described [8 (link),27 (link)–29 (link)]. Briefly, female BALB/c mice purchased from KOATECH (Pyeongtaek, South Korea) were used, where RH and ME49 were maintained via serial intraperitoneal and oral passage, respectively [29 (link)]. Recombinant baculovirus (rBV) and virus-like particles (VLPs) were produced using Spodoptera frugiperda Sf9 cells, which were cultured in spinner flasks at 27°C, 130–140 rpm with SF900 II medium (Invitrogen, Carlsbad, CA, USA). Polyclonal T. gondii antibodies were acquired from sera of T. gondii (ME49)-infected mice. Horseradish peroxidase (HRP)-conjugated secondary antibodies and monoclonal mouse anti-M1 antibody wae purchased from Southern Biotech (Birmingham, AL, USA) and Abcam (Cambridge, UK), respectively.

Serum proteins from patients and a healthy individual were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions and blotted onto nitrocellulose membrane. After blotting, the membrane was blocked with 4% non-fat dried milk, 1% BSA in PBS solution. Then, the membrane was incubated with a polyclonal goat anti-FHR-5 (Cat. number: AF3845, R&D Systems, Minneapolis, Minnesota, US) or monoclonal mouse anti-FHR-5 antibody (clone #390513, Cat. number: MAB3845, R&D Systems, Minneapolis, Minnesota, US) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (rabbit anti-goat and goat anti-mouse, respectively; Southern Biotech, Birmingham, AL, USA). Bound antibodies were detected with Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA). For estimating the molecular weight of the proteins, a protein molecular weight marker containing a mixture of 10 multicolor recombinant proteins was used (Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standard, Bio-Rad, Hercules, CA, USA).