Western blot analysis of extracts from mouse and rat brains using AMPA Receptor 3 (GluA3) (D25G9) 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 3 (GluA3) (D25G9) Rabbit mAb detects endogenous levels of total GluA3 protein. The antibody is not predicted to detect other AMPA receptor subunits (e.g. GluA1, GluA2 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 surrounding His70 of human GluA3 protein.
AMPA- (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), kainite- 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) that assemble as homo- or hetero-tetramers and mediate the majority of fast excitatory transmissions in the CNS. AMPARs are implicated in synapse formation, stabilization and plasticity. Post-transcriptional modifications (alternative splicing and nuclear RNA editing) and post-translational modifications (glycosylation, phoshorylation) result in a very large number of permutations, fine-tuning the kinetic properties of AMPARs (1). GluR 3 knockout mice exhibited normal basal synaptic transmission and long-term depression (LTD) but enhanced long-term potentiation (LTP). In contrast, GluR 2/3 double knockout mice are impaired in basal synaptic transmission (2). Aberrant GluR 3 expression or activity is implicated in a number of diseases, including autoimmune epilepsy, X-linked mental retardation, Rett's syndrome, amyotrophic lateral sclerosis and Alzheimer disease (3).
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