Western blot analysis of total cell lysates from 293 cells, untransfected or transiently transfected with a construct expressing CBP, using Acetyl-β-Catenin (Lys49) Antibody (Top), or total β-Catenin Antibody (Amino-terminal Antigen) #9581 (Bottom).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.
Acetyl-β-Catenin (Lys49) Antibody detects endogenous β-catenin only when acetylated at Lys49.
Mouse, Rat, Pig
Polyclonal antibodies are produced by immunizing animals with a synthetic acetylated peptide corresponding to residues surrounding Lys49 of human β-catenin. Antibodies were 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).
Lys49 lies in a region that contains several Ser/Thr residues whose phosphorylation status regulates the stability of β-catenin and is one of few residues frequently mutated in thyroid anaplastic carcinoma (9). CBP (CREB-binding protein) binds and acetylates β-catenin at Lys49 (10, 11).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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