Western blot analysis of extracts from rat brain, either sham operated or subjected to ischemia (15 min) alone or followed by reperfusion (4 hours), using Phospho-PSD95 (Tyr236/Tyr240) Antibody (upper) and PSD95 Antibody #2507 (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-PSD95 (Tyr236/Tyr240) Antibody detects endogenous levels of PSD95 protein only when phosphorylated at Tyr236 or Tyr240.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr236 and Tyr240 of human PSD95. Antibodies are purified by protein A and peptide affinity chromatography.
Postsynaptic Density protein 95 (PSD95) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins. These family members consist of an amino-terminal variable segment followed by three PDZ domains, an SH3 domain, and an inactive guanylate kinase (GK) domain. PSD95 is a scaffolding protein involved in the assembly and function of the postsynaptic density complex (1-2). PSD95 participates in synaptic targeting of AMPA receptors through an indirect manner involving Stargazin and related transmembrane AMPA receptor regulatory proteins (TARPs) (3). It is implicated in experience-dependent plasticity and plays an indispensable role in learning (4). Mutations in PSD95 are associated with autism (5).
Phospho-PSD95 (Tyr236/Tyr240) Antibody is directed against previously unpublished PSD95 tyrosine phosphorylation sites at Tyr236 and Tyr240 that were identified at Cell Signaling Technology (CST) using PhosphoScan®, CST's MS/MS platform for phosphorylation site discovery. Phosphorylation of PSD95 at Tyr236 and Tyr240 was observed in extracts isolated from ischemic rat brain. The sites were independently found in a large-scale identification of tyrosine phosphorylation sites from murine brain (6).
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