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 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-PSD95 (Tyr236/Tyr240) Antibody detects endogenous levels of PSD95 protein only when phosphorylated at Tyr236 or Tyr240.Species Reactivity:
RatSpecies predicted to react based on 100% sequence homology:
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, a 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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