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 17:28, 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).


Binding Assay 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid (AMPA) receptors
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