Peroxidase conjugated anti rabbit igg

This assay was adapted from the method reported by Karmali et al. [17 (link)]. Briefly, a standard concentration of purified Stx2 (3 μg/10 μL) was resolved into its A and B subunits by SDS-PAGE. Proteins were transferred to nitrocellulose membranes, which were then cut into longitudinal strips, blocked with 0.01 M phosphate buffered saline (PBS), pH 7.2, containing 5% skim milk and 2% bovine serum albumin (BSA) (blocking buffer), and incubated for 2 h with each serum sample diluted 1:250 in blocking buffer. After washes in PBS-0.05% Tween-20, the strips were incubated with a 1:2000 dilution of peroxidase-conjugated anti-human IgG (Sigma, St Louis, MO, USA) in blocking buffer. A polyclonal antibody obtained from a rabbit immunized with Stx2 [25 (link)] was used as the positive control for the assay, and this membrane was incubated with a 1:2000 dilution of peroxidase-conjugated anti-rabbit IgG (Sigma, St Louis, MO, USA) in blocking buffer. Antigen-antibody complexes were visualized with the enhanced chemiluminiscent detection system (ECL, Amersham Life Science), and positive and negative responses were qualitatively analyzed. Each serum sample was assayed at least twice.

Peroxidase conjugated anti-rabbit IgG (Sigma-Aldrich, USA) was used as secondary antibody.

Protein was isolated by acetone precipitation from the cell lysates as previously described20 (link). The protein pellet was dissolved in 1% SDS buffer, warmed for 15 min at 55 °C, and centrifuged for 5 min at 14000 rpm. Protein concentrations in the supernatant were determined using a BCA Protein Assay Kit (Pierce). Proteins were separated on 10% SDS-PAGE gels and transferred to a PVDF membrane followed by Western blot analysis. Briefly, 3% milk in TBS containing 0.1% Tween-20 was used to block non-specific binding. The blot was subsequently incubated with an anti-COX2 rabbit polyclonal antibody (1:500, Santa Cruz) followed by a secondary antibody (peroxidase-conjugated anti-rabbit IgG 1:5000, Sigma). After antibody incubation, blots were extensively washed in TBS containing 0.1% Tween-20. For detection, the ECL kit (Amersham Life Sciences) was used according to the directions of the manufacturer.

MDBK cells were plated on 24-well cell culture plates at 260,000 cells per well and incubated overnight at 37 °C and 5% CO₂. Once cell monolayers were established, 14,000 TCID₅₀ of virus in 200 µL was added to each well of the plate (MOI = 0.01). After 1 h of incubation, final volume was completed up to 1 mL of serum-free DMEM and incubated. After 24 h post-infection, cells were treated with CIGB-325 (30 μM) or vehicle (PBS) in serum-free DMEM for 45 min at 37 °C in 5% CO₂. Subsequently, the culture medium was withdrawn and the cells were washed with PBS, and Western blot was carried out as described (2.7). Primary antibodies against phospho-RPS6 (S235/236) and total RPS6 (Cell Signaling Technology, Danvers, MA, USA) were used, and detection was performed with peroxidase-conjugated anti-rabbit IgG 1:5000 (Sigma, St. Louis, MO, USA).

Cell lysates were separated by SDS/PAGE (8, 10 or 12% polyacrylamide gel), electrotransfered to PVDF membrane (Millipore,) and blocked for 1 h (at room temperature) in 5% non-fat powdered milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (BioShop, Burlington, Canada). Membranes were incubated with primary antibody overnight at 4 °C. After incubation with secondary antibody for 1 h, room temperature, chemiluminescence was detected using Immobilon Western HRP substrate (Millipore) with ChemiDoc system (BioRad). The following antibodies and dilutions were used: rabbit anti-MCPIP1 (1:1000, GeneTex), HIF1α (1:1000), HIF2α (1:1000), VHL (1:500), Akt (1:1000), Phospho-Akt (1:2000), SAPK/JNK (1:500), Phospho-SAPK/JNK (1:1000), p38 (1:1000), Phospho-p38 (1:1000), ERK1/2 (1:1000), Phospho-ERK1/2 (1:1000). All mentioned antibodies were from Cell Signaling Technology. Tubulin (1:4000, Calbiochem; Merck Millipore, Billerica, MA, USA) or in case tissue samples, GAPDH (1:30,000 Sigma-Aldrich) antibodies were used as a loading control. The following secondary antibodies were used: peroxidase-conjugated anti-rabbit IgG (1:30,000; Sigma) and peroxidase-conjugated anti-mouse IgG (1:10,000, Sigma).

Two-dimensional PAGE, isoelectric focusing, was carried out using the protocol described by Gorg et al., 2000 (23 (link)). Protein samples (100 μg of porcine myosin/MLSA) were loaded on IPG strips (Bio-Rad Laboratories, USA) of pH 3–10 for myosin, pH 4–7 for MLSA and length 7 cm. Proteins were separated in second dimension using 10% SDS-PAGE and transferred to nitrocellulose membrane (NCM) (24 (link)). Blotted NCM was blocked with 3% BSA (Sigma, USA) for 1 h, then incubated with pooled leprosy patients’ sera (1:50) while NCM of separated proteins of MLSA was incubated with Myosin-hyperimmunized rabbit sera (1:50). These NCMs were incubated overnight at 4°C followed by three times washing with PBS containing 0.05% Tween-20 and incubated with peroxidase conjugated anti-rabbit IgG (1: 10,000) (Sigma-Aldrich, USA) for 1 h. Later, visualization of antigen antibody reactivity was done by color development with diaminobenzedine (Sigma, USA) solution. Capturing of image was done by Chemidoc (Bio-Rad Laboratories, USA).