Protein (50 mg) obtained from mice brain or intestine was isolated on 10% Tris-Glycine polyacrylamide gels (Bio-rad Laboratories, Inc., Hercules, CA, USA) and transferred to Immobilon-P membranes (Millipore Corporation, Bedform, MA, USA). The membrane was incubated overnight at 4 ℃ with following antibodies: anti-GABAA-a2 (Abcam, #ab72445), anti-GABAB-R1 (Cell signaling, #3835), anti-5HT1A (Abcam, #ab85615), or GAPDH (Cell signaling, #5174). The membrane-bound antibodies were applied to secondary IgG antibody (Cell signaling, #5127) and signals were visualized using ECL Prime (GE Healthcare Life Sciences, Issaquah, WA, USA) on a FluorChem E Imaging System (Protein Simple, San Jose, CA, USA). Protein signals were quantified using FluorChem E Image Quant software with normalization by GAPDH as an internal control.
Anti 5ht1a
Manufactured by Abcam
Sourced in United Kingdom
Anti-5HT1A is a lab equipment product that detects the 5-HT1A receptor, a serotonin receptor subtype. It is used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Tris glycine polyacrylamide gel, by Bio-Rad (1 mentions)
Anti gabab r1, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Goat anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Clarity ecl, by Bio-Rad (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti glun2b, by Abcam (1 mentions)
Secondary antibodies conjugated with horseradish peroxidase hrp, by Santa Cruz Biotechnology (1 mentions)
Anti glua1, by Abcam (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti glun2a, by Abcam (1 mentions)
Anti bdnf, by Abcam (1 mentions)
Chemidox xrs, by Bio-Rad (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
4 protocols using anti 5ht1a
1
Western Blot Analysis of GABA and 5HT1A Receptors
2
Western Blot Analysis of Brain Receptors
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The supernatant from homogenized brain tissue was lysed and 40 µg of protein was separated on 10% SDS-PAGE, then transferred to an Immobilon-P PVDF membrane (Millipore, Burlington, MA, USA). The membrane was blocked with 5% BSA, incubated with primary antibodies overnight at 4 • C, followed by goat anti-rabbit IgG-HRP (1:2000, Cell Signaling Technology, Danvers, MA, USA) for 2 h at room temperature. After three washes with TBST buffer (Dynebio Inc., Seongnam, Republic of Korea), the membrane was visualized using Clarity ECL (Bio-Rad, Hercules, CA, USA) on a UVP GelStudio system (Analytik Jena AG, Jena, Germany). Primary antibodies: anti-GABAA receptor (Abcam, Cambridge, UK; 1:1000), anti-5HT1A (Abcam; 1:1000), anti-GABAB receptor (Cell Signaling Technology; 1:1000), anti-β-actin (Cell Signaling Technology; 1:1000), and goat anti-rabbit IgG-HRP (Cell Signaling Technology; 1:2000).
3
Antibody-based Protein Analysis Techniques
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ZBD-2 was prepared at our laboratory as previously described [13 (link)]. Anti-β-actin antibody was purchased from Sigma (St. Louis, MO). Anti-GluN2A, anti-GluN2B, anti-GluA1, anti-p-GluA1-ser845, anti-5-HT1A and anti-BDNF antibodies were purchased from Abcam (Cambridge, UK). Anti-TSPO, anti-GABAA-α2 and anti-GABAA-γ2 antibodies were purchased from Chemicon (Temecula, CA, USA). All secondary antibodies conjugated with horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The CRH (Corticotropin-releasing hormone), ACTH (Adreno-cortico- tropic-hormone), CORT (Corticosterone) and 5-HT (5-hydroxytryptamine) ELISA kits were purchased from (Cusabio, Wuhan, China). All of the chemicals and reagents used were standard biochemical quality and commercially available.
4
Western Blot Analysis of 5HT1A in Mouse Hippocampus
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Hippocampus of two mice from each group was homogenized in an ice-cold RIPA buffer (Solarbio, China), and centrifuged at 12000×g for 10 min at 4 °C. The supernatants were collected. Protein concentration was determined by a BCA (bicinchoninic acid) protein assay using bovine serum albumin as a standard. The proteins were separated with the aid of sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. After blocking in 3 % bovine serum albumin/Tris buffered saline with Tween 20 (TBST) at room temperature for 2 h, the membranes were incubated with the primary antibody at 4 °C overnight (anti-5HT 1A 1:1000, Abcam, UK). After washing with TBST three times, the membranes were incubated with HRP conjugated anti-rabbit or anti-mouse lgG secondary antibodies (1:10000, Proteintech, USA) for 1 h at room temperature. The blots were washed again three times with TBST buffer, and the immunoreactive bands were detected using a chemiluminescence system (Bio-Rad Chem-iDox XRS, USA). Protein band densities were quantified using the Image J software (Bio-Rad ChemiDoc XRS) while GAPDH (1:5000, Proteintech) was used as a loading control.
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