AMPA Receptor: Difference between revisions
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===Determining ''ad hoc'' experimental parameters for quantitative autoradiography of AMPAR using [<sup>3</sup>H]AMPA ligand=== | |||
===Experimental determination of receptor-ligand binding affinity for [3H]AMPA=== | |||
10 nM [<sup>3</sup>H]AMPA bound 32% of receptors in rat. Frozen-thawed tissue in buffer with KCl bound 36% of receptors. Specific binding curve extrapolation estimates the Kd of AMPA to be ~28 nM ([http://www.sciencedirect.com/science/article/pii/0006899387900308 Olsen et al 1987]). | |||
{| class="mw-collapsible wikitable" style="width: 600px; height: 150px; text-align:center;" border="1" | {| class="mw-collapsible wikitable" style="width: 600px; height: 150px; text-align:center;" border="1" | ||
|+Binding Assay 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid (AMPA) receptors | |+Binding Assay 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid (AMPA) receptors | ||
Revision as of 18:28, 12 April 2012
Determining ad hoc experimental parameters for quantitative autoradiography of AMPAR using [3H]AMPA ligand
Experimental determination of receptor-ligand binding affinity for [3H]AMPA
10 nM [3H]AMPA bound 32% of receptors in rat. Frozen-thawed tissue in buffer with KCl bound 36% of receptors. Specific binding curve extrapolation estimates the Kd of AMPA to be ~28 nM (Olsen et al 1987).
| PROCEDURES | |||
|---|---|---|---|
| Radioligand | Pre-Incubation | Incubation | Wash |
| 3H-AMPA 15 nM |
50 mM Tris-HCl 100 mM KCl pH 7.4 10 min |
15 nM 3H-AMPA Pre-Incubation Buffer 60 min |
Pre-Incubation Buffer 10 min x2 baths 1 dip diH2O |