GABAB receptor constituents revealed by tandem affinity purification from transgenic mice.

저자: Bartoi T, Rigbolt KT, Du D, Köhr G, Blagoev B, Kornau HC.

저널: J Biol Chem. 2010 Jul 2;285(27):20625-33

사용한 제품: Dynabeads Streptavidin MyOne T1 (Cat# 65601, 65602)

Abstract

GABAB receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter  -aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligandbinding subunit GABAB1, are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABAB receptors. The transgenic mice express GABAB1 isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABAB1 complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABAB receptors via the GABAB2 subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABAB2. The mice equipped with tags on GABAB1 facilitate validation and identification of native binding partners of GABAB receptors, providing insight into the molecular mechanisms of synaptic modulation

제품 사용법

Tandem Affinity Purification from NTAP-GABAB1a Mice

40–70 mg of solubilized membrane proteins were prepared from juvenile (P10–P16) mouse brains, and the 2-mercaptoethanol and EDTAconcentrations were adjusted to 5 and 2mM, respectively. The extract was rotated overnight with 720㎕  of streptavidin-coated magnetic beads (Dynabeads MyOne Streptavidin T1, Invitrogen). Afterward, the beads were collected using a magnetic device (Invitrogen) and washed three times with 1 ml of solubilization buffer, and the bound material was eluted with 2 mM biotin in 1600㎕ of solubilization buffer for 20 min on a rotating wheel. The biotin eluate was incubated with 160㎕ of anti-HA affinity matrix (Roche Applied Science) rotating for 4 h. After the beads were washed three times with 1 ml of solubilization buffer, the bound material was eluted in SDS loading buffer (50mM Tris-Cl, pH 8.0, 200mM dithiothreitol, 6% (w/v) SDS, 10% glycerol, 7 M urea, 0.01% (w/v) bromphenol blue) for 10 min at 42 °C, separated on NuPage Novex gels (Invitrogen), and stained with silver nitrate as previously described (34).