Western blot analysis of extracts from rat brain, either sham-operated, 15 min ischemia followed by 4 h reperfusion or 15 min ischemia only, using Phospho-AMPA Receptor 2 (GluA2) (Tyr876) Antibody (upper) or AMPA Receptor (GluA2/3/4) Antibody #2460 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Phospho-AMPA Receptor 2 (GluA2) (Tyr876) Antibody detects endogenous levels of GluA2 only when phosphorylated at Tyr876. It may also detect GluA3 when phosphorylated at the conserved Tyr887. This residue is not conserved in GluA1 or GluA4.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr876 of human AMPA Receptor 2 (GluA2). Antibodies are purified by protein A and peptide affinity chromatography.
AMPA- (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), kainate-, and NMDA- (N-methyl-D-aspartate) receptors are the three main families of ionotropic glutamate-gated ion channels. AMPA receptors (AMPARs) are comprised of four subunits (GluR 1-4), which assemble as homo- or hetero-tetramers to mediate the majority of fast excitatory transmissions in the central nervous system. AMPARs are implicated in synapse formation, stabilization, and plasticity (1). In contrast to GluR 2-containing AMPARs, AMPARs that lack GluR 2 are permeable to calcium (2). Post-transcriptional modifications (alternative splicing, nuclear RNA editing) and post-translational modifications (glycosylation, phosphorylation) result in a very large number of permutations, fine-tuning the kinetic properties of AMPARs. Research studies have implicated activity changes in AMPARs in a variety of diseases including Alzheimer’s, amyotrophic lateral sclerosis (ALS), stroke, and epilepsy (1).
Src family tyrosine kinases phosphorylate the GluR 2 subunit of AMPA receptors at Tyr876, which increases the interaction with GRIP1/2 but not PICK1. In addition, Tyr876 is important for AMPA- and NMDA-induced GluR 2 internalization (3).
The phosphorylation site at Tyr876 was also independently identified at Cell Signaling Technology (CST) using PhosphoScan®, CST's MS/MS platform for phosphorylation site discovery. Phosphorylation of GluR at Tyr876 was observed in extracts isolated from ischemic rat brain.
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