Western blot analysis of extracts from parental (COS) or NMDA Receptor 1 (GluN1)-transfected (COS/GluN1) cells, untreated or forskolin-treated (30 mM for 20 minutes), using Phospho-NMDA Receptor 1 (GluN1) (Ser897) Antibody.
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 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
Phospho-NMDA Receptor 1 (GluN1) (Ser897) Antibody detects transfected NMDA Receptor 1 (GluN1) only when phosphorylated at serine 897.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser897of human NMDA Receptor 1 (GluN1). Antibodies are purified by protein A and peptide affinity chromatography.
N-methyl-D-aspartate receptor (NMDAR) forms a heterodimer of at least one NR1 and one NR2A-D subunit. Multiple receptor isoforms with distinct brain distributions and functional properties arise by selective splicing of the NR1 transcripts and differential expression of the NR2 subunits. NR1 subunits bind the co-agonist glycine and NR2 subunits bind the neurotransmitter glutamate. Activation of the NMDA receptor or opening of the ion channel allows flow of Na+ and Ca2+ ions into the cell, and K+ out of the cell (1). Each subunit has a cytoplasmic domain that can be directly modified by the protein kinase/phosphatase (2). PKC can phosphorylate the NR1 subunit (NMDAR1) of the receptor at Ser890/Ser896, and PKA can phosphorylate NR1 at Ser897 (3). The phosphorylation of NR1 by PKC decreases its affinity for calmodulin, thus preventing the inhibitory effect of calmodulin on NMDAR (4). The phosphorylation of NR1 by PKA probably counteracts the inhibitory effect of calcineurin on the receptor (5). NMDAR mediates long-term potentiation and slow postsynaptic excitation, which play central roles in learning, neurodevelopment, and neuroplasticity (6).
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