Horseradish peroxidase conjugated goat anti rabbit igg antibody

Detection of the proteins by western blotting was performed as described previously [21 (link)]. Equivalent amounts of protein were separated on 12% gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyldifluoride membranes (GE Healthcare, Buckinghamshire, UK). After blocking, the membranes were incubated with primary antibodies: rabbit anti-phospho-GSK-3β (Ser9) (1 : 1000; Cell Signaling Technology, Beverly, MA), rabbit anti-GSK-3β (1 : 1000, Cell Signaling Technology), rabbit anti-phospho-glycogen synthase (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-3 (1 : 1000, Cell Signaling Technology), rabbit anti-cleaved caspase-7 (1 : 1000, Cell Signaling Technology), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 20000; Sigma-Aldrich), at 4°C with gentle shaking overnight. Detection was carried out using horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1 : 3000, Abcam), followed by ECL western blotting detection reagents (GE Healthcare, Buckinghamshire, UK). Western blots were visualized on the Kodak Image Station (Carestream Health Inc., Rochester, NY).

A purified pellet from synchronized schizont stages of the P. falciparum 3D7 strain was separated by SDS-PAGE in a 12% polyacrylamide gel and transferred to nitrocellulose (BioRad). Monoclonal antibodies AbMSP10-1, AbMSP10-2 and AbMSP10-3 at 1 µg/mL were used for the detection of native protein PfMSP10 in schizont parasites lysates. Proteins were detected using a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Abcam, Cambridge, MA, USA). Blots were visualized with FAST DAB (Sigma Aldrich). All antibodies were pre-depleted against non-infected red blood cells before used. Non-infected red blood cells served as a negative control. The revealed band sizes were compared using Precision Plus Protein Dual Color Standards (BioRad) and analysed in Gel Analyzer software 2010a.
The eluted fractions of purified rMSP10 were loaded onto polyacrylamide gels and analysed by western blotting, as already described, using an anti-His antibody (GenScript).

Hippocampal tissue was lysed in ice‐cold radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China), and then centrifuged at 1,000 rpm for 10 min at 4℃. Equivalent amounts of protein (80 μg) were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk (dissolved in triphosphate‐buffered saline with 0.1% Tween (TBST)) for 45 min at room temperature and incubated with primary rabbit antibodies for BDNF (1:800, immunoway), CREB1 (1:1,000, immunoway), p‐CREB1 (1:1,000, immunoway), Synapsin Ⅰ (1:800, abcam), p‐Synapsin Ⅰ (1:800, abcam), PSD‐95 (1:1,500, immunoway), and β‐actin (1:1,000, immunoway), respectively, diluted in blocking buffer at 4℃ overnight. After the membranes were then washed three times in TBST, the horseradish‐peroxidase‐conjugated goat‐anti‐rabbit IgG antibody (1:5,000, abcam) was added to allow incubation for 1 hr at room temperature. The membranes were washed three times in TBST, and the immunoblots were revealed with the chemiluminescent ECL Western Blotting Detection Reagent (Beyotime Biotechnology, China) and visualized by a chemiluminescence system (Tanon 5,200). Optical densities of the bands were scanned and quantified with ImageJ software.

Western blot was performed as described previously [16 (link)]. Briefly, cells were lysed on ice for 5 min in 2 × SDS buffer with β-mercaptoethanol, and were treated in 95 °C for 10 min. Supernatants were harvested by centrifugation for 1 min at 10,000×g. Total proteins were loaded and separated on SDS-PAGE gels, and were electroblotted onto PVDF membrane. The membrane was soaked for 2 h in block solution, and then incubated overnight in the presence of rabbit polyclonal to signal transducers and activators of transcription 3 (STAT3, phospho Y705, ab76315), or STAT3 (ab32500) (Abcam, Cambridge, MA, USA; 1: 1000 dilution). Horseradish peroxidase-conjugated goat anti-rabbit antibody IgG (Abcam; 1: 2000 dilution) was added for additional 2 h incubation. Antigen-antibody complexes were observed by enhanced chemiluminescence (Western Blotting Luminol Reagent, Cell Signaling Technology, Danvers, MA, USA).