Goat anti rabbit igg peroxidase conjugate

Proteins were isolated from gastrocnemius muscles using cOmplete Lysis-M EDTA-free kit (Roche Applied Science). 25 µg of total protein lysate was denatured by boiling in Laemmli buffer and separated using SDS-Page electrophoresis and then, transferred to PVDF membrane (Roche Applied Science). Membranes were blocked with 5 % Blotto (BioRad)/TBS for 1 h and incubated with primary antibodies diluted 1:2000 in 5 % Blotto (BioRad)/TBS, at 4 °C, overnight, followed by secondary antibodies diluted 1:20,000, at room temperature, for 2 h. Protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and exposed to chemiluminescence positive film (Amersham Hyperfilm ECL, GE Healthcare). The following primary antibodies were used: rabbit polyclonal anti-CD68 (Abcam), mouse monoclonal anti-CD163 (AbD Serotec), mouse anti-tubulin (Sigma Aldrich). Secondary antibodies used were: peroxidase-conjugate rabbit anti-mouse IgG (Sigma-Aldrich), peroxidase-conjugate goat anti-rabbit IgG (Sigma-Aldrich). Results were analyzed with Gel Doc™ XR+ System using Image Lab software (BioRad).
All experiments were performed 2–10 times (biological repeats). Results were shown as mean or median, and standard or mean deviations were presented. t test was performed for statistical analysis.

Proteins were isolated from BMSCs, control or transfected with mimic miRNAs, using RIPA buffer (ThermoFisher) supplemented with protease and phosphatase inhibitors. 25 μg of total protein lysates were denatured by boiling in Laemmli buffer, separated using SDS-Page electrophoresis, and transferred to PVDF membranes (Roche). The membranes were blocked with 5% milk/Tris-buffered saline (TBS) for 1h and incubated with primary antibodies diluted 1:1000 in 5% milk/TBS, at 4°C, overnight, followed by secondary antibodies diluted 1:20000, at room temperature, for 2h. Next, protein bands were visualized with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and exposed to chemiluminescence positive film (Amersham Hyperfilm ECL, GE Healthcare). Film was developed in a darkroom using a developer and fixer (Fuji). The density of the examined bands was compared to the density of α-tubulin bands. The following primary antibodies were used: rabbit polyclonal anti-IGFBP2 (Abcam) and mouse monoclonal anti-α-tubulin (Sigma-Aldrich). Secondary antibodies used: peroxidase-conjugated rabbit anti-mouse IgG (Sigma-Aldrich) and peroxidase-conjugate goat anti-rabbit IgG (Sigma-Aldrich). Three independent experiments were performed. The blots were analyzed using Gel Doc XR+ and Image Lab 5.1 (BioRad).

Human APOBEC1 was characterized in intestinal protein extracts by Western blotting. Scrapped intestinal mucosa cells were homogenized with a Dounce homogenizer in RIPA buffer (50 mM Tris-HCl, pH 7.4; 1% IGEPAL; 0.5% Na-deoxycholate; 0.1% SDS; 150 mM NaCl; 2 mM EDTA; 50 mM NaF; 0.2 mM sodium orthovanadate) with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche; 1 mM PMSF; 1 mM Benzamidine) extemporaneously added. After incubation on ice for 30 minutes and centrifugation at 10 000 g for 10 minutes at 4°C, the supernatant was collected and frozen in aliquots at −80°C. Protein concentration was determined by Bradford assay (BioRad, France) using BSA as standard.
Proteins were separated on a 16% acrylamide gel electrophoresis then transferred on Hybond-P membrane. The human APOBEC1 enzyme was detected after incubation with a rabbit anti-APOBEC1 antibody (Sigma, SAB2100132), a goat anti-rabbit IgG peroxidase conjugate (Sigma, A-0545) and the immunofluorescence ECL 2 detection kit (Pierce).

Piscidin1 was examined with an optical microscope and immunohistochemical techniques. Slices were treated with an anti-Piscidin1 antibody overnight in a humid environment. Following a PBS wash, the sections were incubated with a goat anti-rabbit IgG-peroxidase conjugate for 60 min (Sigma-Aldrich, St. Louis, MO, USA, dilution 1:100, source Goat). By allowing the sections to sit in a solution of 0.02% diaminobenzidine (DAB) and 0.015% hydrogen peroxide for 1–5 min at room temperature, the peroxidase activity of the sections was assessed. Sections were dehydrated, mounted, and inspected with a Zeiss Axioskop 2 plus microscope and a Sony Digital Camera DSC-85 after being rinsed in PBS. As a negative control, experiments were conducted without the primary antibody.

The ELISA was conducted as for tick proteins, but plates were coated with 100 ng α-Gal (Galα1-3Gal-BSA, 3 atom spacer, approximately 1.82 × 10²⁰ Gal epitopes/g [37 (link)]; product code NGP0203; Dextra, Shinfield, UK) per well in carbonate/bicarbonate buffer (Sigma-Aldrich), incubated overnight at 4 °C following five washes with PBST. Unspecific unions were blocked with 1% HSA (Sigma-Aldrich) for 1 h at RT. Serum peritoneal fluid samples were diluted (1:100, v/v) in blocking solution, followed by the addition of 100 μl/well and incubation for 1.5 h at 37 °C. Plates were washed three times with PBST, and 100 μl/well of rabbit anti-zebrafish IgM antibodies diluted (1:1,000, v/v) in blocking solution was added and incubated for 1 h at RT. Plates were washed with PBST, and goat anti-rabbit IgG-peroxidase conjugate (Sigma-Aldrich) diluted 1:3000 in blocking solution was added and incubated for 1 h at RT. After washes with PBST, 100 μl/well of TMB (Promega) was added and incubated for 15 min at RT. Reactions were stopped with 50 μl/well of 2N H₂SO₄, and the OD at 450 nm was measured in a spectrophotometer (Multiskan, Thermo Fisher Scientific). Only hemorrhagic type allergic reactions were associated with individual fishes treated with tick saliva, and thus a correlation analysis was conducted between anti-α-Gal IgM antibody titers and these signs in this group (P < 0.05; n = 6).

Soluble extracts of Synechocystis 6803 were prepared as described [31 (link)]. Proteins were separated by Tricine SDS-PAGE [32 (link)] using gels containing 6 M urea and transferred by electrophoresis onto nitrocellulose membranes. Blot membranes were incubated with specific primary antibodies and then with a secondary antibody (goat anti-rabbit IgG-peroxidase conjugate) (Sigma). Immunolabelled bands were visualized using the Immobilon western membrane chemiluminescence system (Millipore, Bedford, MA, USA). For detection of Zn²⁺ -induced fluorescence a 16% Tricine SDS-PAGE without urea containing 1 mM zinc acetate was used.

Primary: rabbit polyclonal anti-CDK/2 (M2), cat no. sc-1632 Santa Cruz Biotechnologies; mouse monoclonal (9E10) anti-c-Myc, cat no. SC-40 Santa Cruz Biotechnologies; rabbit polyclonal anti-p-c-Myc (Thr58/Ser 62), cat no. Sc-8000R, Santa Cruz Biotechnologies; mouse monoclonal anti-SCML2 (SCMAD14a), cat no. ab51506 Abcam; rabbit polyclonal antip-Src (Tyr416), cat no. 2101S, Cell Signaling; rabbit polyclonal anti-Src antibody, cat no. 2108S, Cell Signaling, rabbit monoclonal anti-p27 Kip1 (D69C12) XP^® cat no. 3686; Santa Cruz Biotechnology; mouse monoclonal p21 (F-5), cat no. sc-6246, Santa Cruz Biotechnologies; mouse monoclonal anti Nanog, clone 7F7-1, cat no. MABD24, EMD Millipore; rabbit polyclonal anti-Bmi-1 antibody, cat no. ab38295, Abcam. Mouse monoclonal anti-tubulin, cat no. T5168, Sigma. Secondary: anti-mouse IgG (A9044; Sigma-Aldrich); and goat anti-rabbit IgG peroxidase conjugate (A0545; Sigma-Aldrich).