Western Blotting Protocol with Gene Name: HTR1A Antibody (Catalog Number: AVARP13041_P050)
Catalog Number: AVARP13041_P050
1. Cordeaux Y, Pasupathy D, Bacon J, Charnock-Jones DS, Smith GC. Characterization of serotonin receptors in pregnant human myometrium. J Pharmacol Exp Ther. 2009 Mar,328(3):682-91. Epub 2008 Dec 15. PubMed PMID: 19075042.
Product Name: HTR1A antibody - N-terminal region (AVARP13041_P050)
Gene Name: HTR1A
Species: Human myometrial cells
Experiment Name: Western Blotting
1. Yolande et al. studied the effects of a range of 5-HT receptor subtype-selective agonists and antagonists in isolated strips of myometrium obtained at the time of caesarean section. Candidate receptors were then further studied using RT-PCR, radioligand binding, Western blot, and immunohistochemistry.
2. Three antibodies raised against the human 5-HT1A receptor were used to detect the protein in human myometrium using Western blotting. A major band corresponding to a molecular mass of 35 kDa was observed with all three antibodies (Santa Cruz Biotechnology, Inc., Aviva Systems Biology, and Everest Biotech) in six samples of myometrium, from separate donors.
1. Protein samples were denatured by heating in Tris-glycine SDS sample buffer supplemented with dithiothreitol (NuPAGE reducing agent, Invitrogen) at 85 C for 2 min.
2. Proteins were separated by SDS-polyacrylamide gel electrophoresis (125 V for 2 h) on 10 or 12% Tris-glycine gels (Invitrogen). Proteins were then transferred to polyvinylidene difluoride membranes (Invitrogen) using an X-Cell II Blot Module (Invitrogen), according to the manufacturer's instructions (25 V, 2 h).
3. After transfer, membranes were incubated in 5% milk/Tris-buffered saline/Tween 20 solution for 2 h at room temperature to reduce nonspecific binding.
4. Membranes were incubated with primary antibodies in 5% milk/TBST solution at 4 C overnight. Membranes were washed in TBST solution (12 × 5-min washes), before incubation with the respective secondary antibody immunoglobulin horseradish peroxidase conjugate (5% milk/TBST solution) for 1 h at room temperature.
5. After a further series of washes with TBST solution (as above), membranes were incubated with Enhanced Chemiluminescence (ECL Plus) detection reagents (5 min, room temperature). Membranes were then wrapped in cling film and exposed to Hyperfilm-ECL X-ray film for up to 5 min.
Other reagents used: NaCl, KCl, MgSO4, NaHCO3, KH2PO4, CaCl2, and D-glucose.