Western blot analysis of extracts from mouse and rat brains using AMPA Receptor 2 (GluA2) (D39F2) Rabbit mAb.
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. 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 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
AMPA Receptor 2 (GluA2) (D39F2) Rabbit mAb detects endogenous levels of total GluA2 protein. The antibody is not predicted to recognize other AMPA receptor subunits (e.g. GluA1, GluA3 or GluA4) based on sequence homology of the antigen.Species Reactivity:
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human GluA2 protein.
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 (4). Phosphorylation of GluR 2 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 (5).
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