Goat anti rabbit igg hrp

Crude LPS extracts were prepared as described (Davies et al., 2013 (link)). After running the samples on TSDS–PAGE (Tricine-SDS–PAGE) gels, two methods were used for visualization: either silver stain as described previously (Davies et al., 2013 (link)) or Western blot. For Western blots, the samples were transferred to polyvinylidene fluoride (PVDF) and blocked with phosphate buffered saline with 0.1% Tween-20 (PBS-T) and 5% milk. The O4 serum from Statens Serum Institute (Denmark) was used as primary antibody followed by goat anti-rabbit IgG-HRP from Sigma (A0545). Millipore's Luminato Western HRP substrate was used for detection.

The 96-well plates (Corning, Glendale, AZ, USA) were coated with 100 µL of antigen (1 µg/mL) in 2 M urea in PBS overnight at 4 °C. The plates were then washed with PBST (PBS containing 0.05% Tween-20) and blocked with a blocking buffer (PBST containing 1% of casein) for 1.5 h at RT. Convalescent and healthy donor sera, immunized animal sera, or IgY preparations were serially diluted and added to the blocked plates. After incubation at RT for 1 h, the plates were washed three times with PBST and incubated with goat anti-human IgG-HRP (GenScript Piscataway, NJ, USA); goat anti-mouse IgG-HRP (Sigma–Aldrich, St. Louis, MO, USA); goat anti-rabbit IgG-HRP (Sigma–Aldrich, St. Louis, MO, USA); anti-hamster IgG-HRP (Invitrogen, Carlsbad, CA, USA); goat anti-ferret IgG, IgA, and IgM-HRP (Sigma-Aldrich, St. Louis, MO, USA); or anti-IgY-HRP (1:20,000) (Thermo Fisher Scientific, Carlsbad, CA, USA) secondary antibodies at RT for 1 h. The plates were washed three times with PBST, and the HRP substrate TMB (Amresco, Solon, OH, USA) was added. The reactions were stopped using 1N HCl. The absorbance was measured at 450 nm using a Varioskan Lux multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).
The endpoint titer for each serum was defined as the maximum dilution at which a positive result was obtained (>median + 3 × SD of the ODs of the negative controls).

Equal amounts of lysates (20 µL) were separated on a 10% Tris-HCL SDS-PAGE and transferred to a nitrocellulose membrane. Membrane was incubated with rabbit anti-RLuc (Biomol, Hamburg, Germany) as first antibody and goat anti-rabbit-IgG-HRP (Sigma Aldrich, Taufkirchen, Germany) as secondary antibody. Rabbit anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (R&D Systems, Wiesbaden, Germany) in combination with goat anti-rabbit-IgG-HRP was used as loading control.

Western blotting was performed using the Thermo scientific miniblot module. SDS-PAGE of reduced samples was run on Bolt Bis-Tris 4–12% premade gels in MOPS. Blotting was performed onto 0.2nm nitrocellulose membranes at 15V for 30 minutes. Blots were blocked with 5% milk in PBS-0.05% Tween 20 for 1h at room temperature, then incubated overnight at 4°C with primary antibodies at 1:2,000: rabbit anti-PA polyclonal (Genetex), mouse anti-β-actin (Sigma, clone AC-74), and mouse anti-influenza NP (Kerafast, clone HT103). Secondary antibodies (1:3,000) goat anti-Rabbit IgG-HRP (Sigma) and goat anti-mouse IgG-HRP (Promega) were incubated with the blots for 1h at room temperature and the blots were developed using Bio-Rad Clarity Western ECL Substrate. Images were taken on the Bio-Rad ChemiDoc MP imaging system.

Whole cells were lysed as described [42 (link)], and resolved by SDS-PAGE, followed by Western blotting with PVDF membranes (Millipore, Burlington, MA, USA). The filters were probed with the following antibodies: polyclonal anti-VE Cadherin (Bioss Antibodies, Woburn, MA, USA), polyclonal anti-αV (Millipore), polyclonal anti-GAPDH (Elabscience, Houston, TX, USA), and polyclonal anti-actin (Sigma-Aldrich, St. Louis, MO, USA). Then, goat anti-rabbit IgG-HRP (Sigma) or goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA) were employed as secondary antibodies. The peroxidase activity was revealed with Immobilon^TM Western HPR Substrate (Millipore). Films were imaged using the CanoScan 4400F (Canon, Tokyo, Japan) at 300 dpi, with the Adobe Photoshop Creative Suite 2 or CS2, and bands were quantified with the ImageJ 1.52a software (NIH, Bethesda, MD, USA).

Cells were lysed in RIPA buffer (Boston Bioproducts) supplemented with protease inhibitors (Complete, EDTA-free; Roche Diagnostics) and PMSF (Calbiochem). Lysates were separated on a 12% acrylamide gel and subjected to Western blot analysis. Immunoblots were incubated over night at 4°C with the following primary antibodies: anti-RBAK (mouse monoclonal; MBL; 1:2, 000), anti-MMP11 (Rabbit polyclonal; Cell Signaling Technologies; 1:1, 000), anti-pro-caspase 3, P17 (Proteintech, UK), and anti-Actin antibodies (goat polyclonal ; Santa Cruz Biotechnology; 1:2, 000). Goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP (Sigma–Aldrich, USA) secondary antibodies were used to visualize bands using Amersham ECL Prime (GE Healthcare, UK). Signal intensity of Western blots was quantified by Quantity One Software (Bio-Rad, USA).