Horseradish peroxidase hrp conjugated anti mouse igg antibody

The following materials were commercially obtained from the sources indicated: Dexamethasone, p-nitrophenyl-β-d-2-acetoamide-2-deoxyglucopyranoside, compound 48/80, an anti-dinitrophenyl IgE antibody (clone SPE-7), N-succinyl-Ala-Ala-Pro-Phe-pNA, mitomycin C, substance P, and dinitrophenyl human serum albumin (DNP-HSA) from Sigma-Aldrich (St. Louis, MO, USA), Toluidine blue, Safranin-O, and Evans blue from Wako Pure Chemical Industries (Osaka, Japan), an anti-trinitrophenyl IgE antibody (clone IgE-3) from BD Biosciences (San Diego, CA, USA), trinitrophenyl bovine serum albumin (TNP-BSA) from LSL (Tokyo, Japan), H-d-Ile-Pro-Arg-pNA (S-2288) from Chromogenix (Milano, Italy), N-(4-Methoxyphenylazoformyl)-Phe-OH potassium salt (M-2245) from Bachem AG (Bubendorf, Switzerland), an anti-G_αi2 antibody from Santa Cruz Biotechnology (Dallas, TX, USA), an anti-pan-actin antibody (clone C4), an anti-G_αi1 antibody, an anti-G_αi3 antibody, and thapsigargin from Merck Millipore (Billerica, MA, USA), a horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody and a HRP-conjugated anti-rabbit IgG antibody from Agilent Technologies (Santa Clara, CA, USA) and recombinant mouse IL-3 from R & D Systems (Minneapolis, MN, USA). All other chemicals were commercial products of reagent grade.

After terminal anesthesia, blood was collected by cardiac puncture into tubes containing heparin at week 4 (day 30 after immunization), 8 (day 60 after immunization), and 15 (day 100 after immunization) (Figure 1C). Blood samples were centrifuged at 4°C, and plasma samples were stored at −80°C.
The presence of nAChR-binding antibodies was determined using dot blot analysis. Nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) were spotted with a 1.0 μg/μL RIPA) buffer solution of P1 or P2 peptide (1 μL/spot). After drying, the membranes were blocked with 5% non-fat dry milk in PBS for 1 h at room temperature and then washed thrice in PBS with 0.1% Tween (PBS-T). The membranes were incubated with a 1:100 dilution of mouse serum in 5% non-fat dry milk/PBS overnight at 4°C. After washing thrice with PBS-T, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Agilent Technologies, Santa Clara, CA, USA) at a 1:2000 dilution for 1 h at 4°C. Thereafter, the membranes were washed three times and incubated with ECL™ Prime Western Blotting System (GE Healthcare Life Sciences, Chicago, IL, USA) for 5 min. The light intensity of spots on the membrane was detected using a ChemiDoc Touch Imaging System (Bio-Rad).