Western blot analysis of extracts from HEK293 cells untreated and treated with calyculin A (50 nM for 30 minutes), using Phospho-β-Catenin (upper) and β-Catenin Antibody #9562 (lower).Learn more about how we get our images.
Western blot analysis of extracts from HEK293 cells untreated and treated with 50 nM calyculin A for 30 minutes, using Phospho-β-Catenin (Ser45) Antibody (lanes 1-2) or the same antibody after preincubation with listed peptides (lanes 3-8). (S45-P is phospho-Ser45 peptide; S33/37/T41-P is phospho-Ser33/37/Thr41 peptide; S45-non-P is nonphospho-Ser45 peptide.)Learn more about how we get our images.
Western blot analysis of extracts from SW480 cells, using Phospho-β-Catenin (Ser45) Antibody.Learn more about how we get our images.
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
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.
Phospho-β-Catenin (Ser45) Antibody detects endogenous levels of beta-catenin only when phosphorylated at serine 45. It does not recognize β-catenin phosphorylated at other sites.
Mouse, Rat, Dog, Pig
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser45 of human β-catenin. Antibodies are purified by protein A and peptide affinity chromatography.
β-Catenin is a key downstream effector in the Wnt signaling pathway (1). It is implicated in two major biological processes in vertebrates: early embryonic development (2) and tumorigenesis (3). CK1 phosphorylates β-catenin at Ser45. This phosphorylation event primes β-catenin for subsequent phosphorylation by GSK-3β (4-6). GSK-3β destabilizes β-catenin by phosphorylating it at Ser33, Ser37, and Thr41 (7). Mutations at these sites result in the stabilization of β-catenin protein levels and have been found in many tumor cell lines (8).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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|9564S||100 µl||$ 303.0|