Five rats in each group were randomly selected and sacrificed after intraperitoneal injection of 4% chloral hydrate (1 ml/100 g). The isolated hippocampus tissues were quickly transferred to pre-labeled EP tubes precooled on dry ice. The Western blot method has been described previously (n = 5/group, P34) (25 (link)). Briefly, after blocking, the polyvinylidene fluoride membrane blots were incubated with one of the following antibodies: goat anti-ZnT3 polyclonal antibody (1:100, Santa Cruz), goat anti-ZnT4 polyclonal antibody (1:100, Santa Cruz), goat anti-ZIP7 polyclonal antibody (1:100, Santa Cruz), rabbit anti-PHB polyclonal antibody (1:2000, Abcam), rabbit anti-PINK1 polyclonal antibody (1:1000, Abcam), mouse anti-DRP1 monoclonal antibody (1:500, Abcam), rabbit anti-Cathepsin E polyclonal antibody (1:1000, Abcam), rabbit anti-CaMKIIα polyclonal antibody (1:2000, Abcam), rabbit anti-Leptin polyclonal antibody (1:2000, Abcam) or rabbit anti-GAPDH polyclonal antibody (1:5000, Beyotime Biotechnology) in TBST contain 5% nonfat dry milk overnight at 4°C. After washing with TBST 3 times, the blot was incubated with the secondary antibody for approximately 2 h at ambient temperature. The antibody reactions were exposed with Kodak X-ray film using the ECL assay system, Amersham imager 600 (GE Healthcare, USA). The intensity of each band was analyzed by Image Pro Plus (IPP) software.
Rabbit anti gapdh polyclonal antibody
Manufactured by Beyotime
Sourced in China
Rabbit anti-GAPDH polyclonal antibody is a primary antibody that recognizes the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein from a variety of species. GAPDH is a widely expressed and commonly used housekeeping protein.
Automatically generated - may contain errors
5 protocols using rabbit anti gapdh polyclonal antibody
1
Hippocampal Protein Expression Analysis
2
Western Blot Analysis of GABRD
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Total cellular protein extraction and western blotting were performed, as previously described [37 (link)]. The following antibodies were used: a rabbit anti-GABRD polyclonal antibody (1:1,000 dilution; abs141150; Absin Bioscience Inc., Shanghai, China) and a rabbit anti-GAPDH polyclonal antibody (1:2,000 dilution; Beyotime Biotechnology, Shanghai, China) as the loading control.
3
Protein Extraction and Western Blotting
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Western blotting and total cellular protein extraction were carried out (23 (link)). The antibodies including: a rabbit anti-GABRD polyclonal antibody (abs141150; 1:1,000 dilution;), which was from Absin Bioscience Inc., Shanghai, China, and a rabbit anti-GAPDH polyclonal antibody (1:2,000 dilution), which was from Beyotime Biotechnology, Shanghai, China, as the loading control.
4
Western Blot Analysis of Signaling Pathways
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RP‐2 cells were cultured for 45 minutes or 12 hour or 24 hour in the absence or presence of stimulators, respectively. The cells were alternatively pre‐treated with Bay 11 or ST2825 for 30 minutes or Si‐RNA for 48 hour. Total cell protein or nuclear protein or cytoplasmic protein was extracted from the cells using, respectively, an Animal Cell Active Protein Extraction Kit, or a Nuclear and Cytoplasmic Protein Extraction Kit (Sangon Biotech, Shanghai, China). Protein samples (20 μg each) from RP‐2 cells or pancreatic tissue samples were separated on 10% SDS‐PAGE gels, and transferred to nitrocellulose. After washing with Tris buffer saline containing 0.1% Tween 20 (TBS/T) and blocking with 2.5% non‐fat milk, the membranes were separately incubated at 4°C overnight with rabbit polyclonal anti‐TLR4 (Boster, Wuhan, China), anti‐col1, anti‐BAMBI, anti‐α‐SMA (Protein Tech, USA), anti‐IkBα, anti‐pIkBα, anti‐p65, anti‐pSmad2, anti‐pSmad3, anti‐Smad1/2/3 antibodies, goat polyclonal anti‐p50 (Santa Cruz, USA), rabbit polyclonal anti‐pERK1/2, anti‐p‐p38 antibodies (Protein Tech, USA), rabbit polyclonal anti‐GAPDH antibody (Beyotime, Shanghai, China). Blots were incubated for 1 hour with HRP‐linked goat anti‐rabbit IgG or HRP‐labelled Donkey Anti‐Goat IgG (Beyotime) and washed extensively with TBS/T before detection using the ECL system (Thermo, USA).
5
Immunoblotting Analysis of Signaling Pathways
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Protein was extracted from thymus homogenates and subjected to SDS-PAGE gel electrophoresis. The bands were incubated with rabbit polyclonal anti-NF-κB antibody (1:1000, Wanleibio, China), rabbit polyclonal anti-Sirt1 antibody (1:1000, Wanleibio, China), mouse monoclonal anti-AMPK 1 antibody (1:1000, Bioss, China), rabbit polyclonal anti-pAMPK antibody (1:1000, CST) and rabbit polyclonal anti-GAPDH antibody (1:2000, Beyotime, China). Finally, the ECL chromogenic agent was added to expose the image, and the images were analyzed by Image J.
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