Horseradish peroxidase hrp conjugated goat anti mouse igg

pPG-E-α-β2-ε-β1/L. crispatus N-11 was cultured in MRS medium at 37 °C for 16 h, centrifuged at 10,000× g for 5 min, and washed three times with sterilized phosphate-buffered saline (PBS; pH = 8.0). The precipitate was incubated with lysozyme and ultrasonicated. Then, 2 × sodium dodecyl sulfate (SDS) buffer solution was added and the sample was boiled for 10 min. After boiling, the sample was centrifuged, and the supernatant was collected. The supernatant was then subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in a 10% gel, followed by western blot analysis using an anti-α toxin polyclonal and mouse anti-toxoid monoclonal antibody (generated in our laboratory) at a dilution ratio of 1:500. Subsequently, the membrane was incubated with the secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma, USA) diluted 1:5000. Immunolabeled bands were visualized using chemiluminescent substrate reagent (Pierce, Rockford, IL, USA).
To further analyze the protein expressed on the cell surface of pPG-T7g10-PPT/L. crispatus N-11, the recombinant pPG-E-α-β2-ε-β1/L. crispatus N-11 was cultured in MRS medium at 37 °C for 16 h, centrifuged at 10,000× g for 5 min, washed three times with sterilized PBS, resuspended in sterile PBS, and then the cells were examined using laser confocal microscopy [19 (link)].

1 OD₆₀₀ (1 OD₆₀₀ ≈ 10⁷ cells) equivalent recombinant yeasts were collected after induction for Western blot analysis as described elsewhere [32 (link)–34 (link)]. A monoclonal mouse anti-HA (H5N1 A/Vietnam/1203/2004) antibody NR-2729 (BEI Resources, Manassas, VA) (1 : 500 dilution) and horseradish peroxidase- (HRP-) conjugated goat anti-mouse IgG (1 : 5000 dilution) (Sigma-Aldrich Co., St. Louis, MO) were used as primary and secondary antibodies.

Baby hamster kidney cells (BHK-21 clone 15) [28 (link)], Aedes albopictus mosquito cells (C6/36) [29 (link)], dengue virus prototypes DENV-1 (Hawaii), DENV-2 New Guinea C (NGC), DENV-3 (H87) and DENV-4 (H241) were kind gifts from Professor John Aaskov, WHO collaborating Centre for Arbovirus Reference and Research, Queensland University of Technology, Australia.
Stocks of DENV prototype viral strains were grown using confluent monolayer of C6/36 cells. Plaque titration was performed on BHK-21 cells as previously described by Morens et al. (1985) with modifications. Briefly, cell monolayers were prepared by seeding 24 well tissue culture plates and infected with serial ten-fold dilutions (10⁻¹ to 10⁻⁶) of virus in RPMI-1640 (Invitrogen, MA, USA) containing 2% heat-inactivated FBS (Invitrogen, MA, USA). After 2 hours, the wells were overlaid with 1.5% w/v CMC overlay medium and incubated at 37°C with 5% CO₂ for 5–6 days. The infected cells positive for the E protein of DENV were enumerated by intracellular staining using the monoclonal antibody 4G2 (clone D1-4G2-4-15) (Chemicon, Merck Millipore, MA, USA) which was recognized by Horseradish Peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma, MO, USA).

Methyl-β-cyclodextrin (MβCD), cholesterol, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole (DAPI) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Shanghai, China). Filipin III was purchased from Cayman (Michigan, United States). A mouse monoclonal antibody against σC of ARV and polyclonal antibodies against μNS and p10 of ARV were prepared by our laboratory (Duan et al., 2015 (link); Niu et al., 2019 (link)). A rabbit monoclonal antibody against Caveolin-1 was purchased from Cell Signaling Technology (Massachusetts, United States). A mouse monoclonal antibody against β-Actin and secondary antibodies, including fluorescein (FITC)-conjugated goat anti-mouse/rabbit IgG, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, and cholera toxin subunit B (CTB) conjugated to Alexa Fluor^® 594 (CTB- Alexa Fluor 594), were purchased from Sigma-Aldrich. RIPA lysis buffer, PMSF, and SDS-PAGE loading buffer were purchased from Beyotime Biotechnology (Shanghai, China). The chemiluminescent substrate and DRAQ5 were purchased from Thermo Fisher Scientific (Shanghai, China).

Citrinin (CTN), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-(morpholino) ethanesulfonic acid (MES), 2-mercaptoethanol, methanol, bovine serum albumin (BSA), ovalbumin (OVA), polyethylene glycol, 1450 (PEG 1450), Freund’s complete adjuvant, Freund’s incomplete adjuvant, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, hypoxantine, aminopterin and thymidine (HAT) medium, hypoxanthine-thymidine (HT) medium, mouse monoclonal antibody isotyping reagent (IgG1, IgG2a, IgG3, IgM, IgA), and RPMI 1640 were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). The Sp2/0 myeloma cell was stored within our laboratory in liquid nitrogen. Female Balb/C mice (8-weeks old) were purchased from the Wushi animal laboratory (Shanghai, China). All other reagents were chemical grade and obtained from commercial sources in China.

Here, ELISAs were used to screen recombinant proteins (diluted 10 μg/ml in PBST) for ability to attach to individual ECM components using a previously described method [71 ]. All ECM macromolecules were from Sigma-Aldrich (Dorset, UK): collagen I from bovine skin, elastin from bovine neck filament, heparan sulfate from bovine kidney, chondroitin sulfate from bovine cartilage, and laminin-1 from basement membrane mouse sarcoma. Recombinant proteins were added to microplate wells coated with individual ECM components. Bound recombinant proteins were detected using primary antibody, mouse anti-polyhistidine IgG antibodies (Sigma-Aldrich, Dorset, UK) and secondary antibody, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma-Aldrich, Dorset, UK). TMB substrate was added, then 0.5M HCl to terminate colorimetric reaction and OD read at 450nm. Statistical analysis compared the ELISA ODs for the negative control, BSA, with those for components of the ligand panel, using One-way ANOVA and Dunnett’s Multiple Comparison Test.

General reagents were obtained from Sigma-Aldrich (Gillingham, Dorset, UK) or BDH-Merck (Poole, Dorset, UK) unless specified otherwise. Oligonucleotide primers were supplied by Tri-I Biotech (Taipei, Taiwan). DNA and protein reagents were obtained from Invitrogen (Carlsbad, CA), Qiagen (Valencia, CA), Fermentas (ON, Canada), New England Biolabs (MA, USA), or Amersham (GE Healthcare). Antibodies (Abs) used in the study are: EMR2 stalk-specific 2A1 from AbD Serotec (Kidlington, UK); Anti-CD71 (H68.4) from Zymed Laboratories (S. Francisco, CA, USA); Anti-β-actin (clone C4) and Chicken anti-DAF (CD55) polyclonal Abs from Chemicon (California, USA); Fluorochrome-conjugated goat anti-mouse IgG was from Jackson ImmunoResearch (West Grove, PA, USA); Mouse IgG₁ isotype control (clone 11711) was from R&D Systems (MN, USA); Anti-Myc was from Invitrogen; Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, anti-G_αi1,2 and anti-G_β were from Sigma-Aldrich; Anti-EMR2/CTF-subunit polyclonal Ab has been described previously^{37 (link)}.