Western blot analysis of extracts from rat brain, either 15 min ischemia followed by 4 h reperfusion or 15 min ischemia only, using Phospho-AMPA Receptor 2 (GluA2) (Tyr869/Tyr873/Tyr876) Antibody (upper) or AMPA Receptor (GluA2/3/4) Antibody #2460 (lower).Learn more about how we get our images.
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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
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.
Phospho-AMPA Receptor 2 (GluA2) (Tyr869/Tyr873/Tyr876) Antibody detects endogenous levels of GluA2 only when phosphorylated at Tyr869, Tyr873 or Tyr876. It may also detect GluA3 when phosphorylated at the conserved Tyr880, Tyr884 or Tyr887. These residues are not conserved in GluA1 or GluA4.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr869, Tyr873 and 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 sites at Tyr869, Tyr873 and Tyr876 were identified at Cell Signaling Technology (CST) using PhosphoScan®, CST's MS/MS platform for phosphorylation site discovery. Phosphorylation of GluR2 at Tyr869, Tyr873 and Tyr876 was observed in extracts isolated from ischemic rat brain. These sites were independently found in a large-scale identification of tyrosine phosphorylation sites from murine brain (4).
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