Recently our lab became interested in high-affinity receptor binding using 3H-epibatidine from PerkinElmer. We are thinking of using a single known saturating concentration of 2 nM for each sample homogenate. We are using small amounts of tissue but should be able to use a tritiated radioligand based on previous reports.
If anyone has a protocol for high-affinity nAChR binding or related assays using 3H-epibatidine (or similar radioligands; i.e. 125I-epibatidine), that information would be much appreciated and will not go unnoticed.
Thanks.
Yes you can. I would suggest to use Perkin Elmer's WGA SPA Beads to do this. Using 3H-Epibatidine and the beads you can do whole cell binding assays without homogenizing the cells. The Scintillation Proximity Assay (SPA) beads will act as the scintillant when in proximity to the 3H-ligand. I used a microbeta scintillation plate reader to measure counts. You should first optimize for the cell number and bead amount which will vary for each cell line and receptor depending on the concentration of receptors on the surface of the cells. Once you have the cell number and bead amount optimized you can then do a saturation curve or direct titration of 3H-epibatidine to identify the true kd and then determine the best concentration to use in your future experiments.
I was able to develop a decent protocol for overexpressed nicotinic receptors in HEK293 cells by using this paper as reference:
HUI, X., GAO, J., XIE, X., SUTO, N., OGIKU, T. and WANG, M.-W. (2005), A robust homogeneous binding assay for α4β2 nicotinic acetylcholine receptor. Acta Pharmacologica Sinica, 26: 1175–1180. doi: 10.1111/j.1745-7254.2005.00202.x PMID: 16174432
Hope this helps and Good luck!
John
Try: Maritt A M et al., Nicotinic cholinergic receptors in the rat retina: simple and mixed heteromeric subtypes. J Pharm Exp. There 68(2005): 1656-1668.
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