Anti rabbit horseradish peroxidase hrp conjugate

The SDS-Page and Western blot were used to detect expression of Hcp in the mono-species biofilm on the blood agar plates as described previously.⁵⁸ (link)^,59 (link) For the Western blot, we used a rabbit polyclonal antiserum specific for V. cholerae Hcp³⁷ (link) (final dilution 1:5,000). The Hcp proteins of V. cholerae and A. aphrophilus exhibit approximately 70 % amino acid sequence identity. As a secondary antibody, anti-rabbit horseradish peroxidase (HRP)-conjugate was used (Jackson ImmunoResearch, Newmarket, UK) (1:10,000). Immunoreactive bands were visualized using Clarity™ Western ECL Substrate (Bio-Rad) and the ChemiDoc™ XRS+ System (Bio-Rad).

Polyclonal rabbit antiserum against CV-A9 and HPeV-1 were from laboratory collections [25 (link),26 (link),28 (link)]. Alexa Fluor 488-labeled anti-rabbit secondary antibody and nuclei stain DAPI were from Life Technologies. In in vitro assay was used anti-rabbit horseradish peroxidase (HRP) conjugate (Jackson ImmunoResearch).

First 25 μg of total protein isolated with PB buffer (60 mM Tris-base, 2% SDS, 10% sucrose, 2 mM PMSF) was run on NuPAGE 3%–8% Tris-Acetate gels (Thermo Fisher Scientific) in Tris-Acetate SDS Running Buffer (Life Technologies) at 4 °C. The immunoreaction was performed using the following antibodies: anti-ataxin-7 (NBP1-42657, Novus Biologicals, Littleton, CO, USA), anti-vinculin (4650, Cell Signaling Technology, Danvers, MA, USA), and anti-rabbit horseradish peroxidase (HRP)-conjugate (Jackson ImmunoResearch, West Grove, PA, USA,) and detected using WesternBright Quantum HRP Substrate (Advansta, Menlo Park, CA, USA). The protein bands were scanned directly from the membrane using a camera and were quantified using the Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA).