Revision 7
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IHC-P, IF-IC, FC-FP, ChIP, ChIP-seq, C&R

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

92

Source/Isotype:

Rabbit IgG

UniProt ID:

#P35222

Entrez-Gene Id:

1499

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Immunohistochemistry (Paraffin) 1:400 - 1:1600
Immunofluorescence (Immunocytochemistry) 1:800 - 1:3200
Flow Cytometry (Fixed/Permeabilized) 1:800 - 1:1600
Chromatin IP 1:100
Chromatin IP-seq 1:100
CUT&RUN 1:100

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

For a carrier free (BSA and azide free) version of this product see product #33893.

Specificity / Sensitivity

Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb recognizes endogenous β-catenin protein when residues Ser33, Ser37, and Thr41 are not phosphorylated. It does not detect β-catenin protein if tri-phosphorylated at Ser33/Ser37/Thr41. This antibody may also detect β-catenin protein when singly phosphorylated at Ser33. This specificity data was derived from competition ELISA and dot blot analysis using synthetic peptides.

Species Reactivity:

Human, Mouse, Rat, Monkey

Species predicted to react based on 100% sequence homology

Chicken, Xenopus, Zebrafish, Bovine, Dog, Pig, Horse, Guinea Pig

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser37 of human β-catenin protein.

Background

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).

Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb is designed to specifically recognize the stabilized form of β-catenin, i.e., protein that has not been phosphorylated by GSK-3, and thus is functionally active in cell-cell adhesion and/or the canonical Wnt signaling pathway.

  1. Cadigan, K.M. and Nusse, R. (1997) Genes Dev 11, 3286-3305.
  2. Wodarz, A. and Nusse, R. (1998) Annu Rev Cell Dev Biol 14, 59-88.
  3. Polakis, P. (1999) Curr Opin Genet Dev 9, 15-21.
  4. Amit, S. et al. (2002) Genes Dev 16, 1066-76.
  5. Liu, C. et al. (2002) Cell 108, 837-47.
  6. Yanagawa, S. et al. (2002) EMBO J 21, 1733-42.
  7. Yost, C. et al. (1996) Genes Dev 10, 1443-54.
  8. Morin, P.J. et al. (1997) Science 275, 1787-90.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: 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.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalStain is a registered trademark of Cell Signaling Technology, Inc.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 7
#8814

Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb

Western Blotting Image 1: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
Western blot analysis of extracts from control HeLa cells (lane 1) or β-Catenin knockout HeLa cells (lane 2) using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in the β-Catenin knockout HeLa cells confirms the specificity of the antibody for β-Catenin.
Western Blotting Image 2: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
Western blot analysis of extracts from 293T cells, untreated (-) or treated (+) with LiCl (20 mM, 20 hr at 37ºC), using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb (left) and β-Catenin Antibody #9562 (right). Equal protein loading was confirmed using β-Tubulin (9F3) Rabbit mAb #2128.
Western Blotting Image 3: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
Western blot analysis of extracts from 293T cells, untreated (-) or treated (+) with MG132 (10 μM, 4 hr at 37ºC), using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb (left), Phospho-β-Catenin (Ser33/37/Thr41) Antibody #9561 (center), or β-Catenin (6B3) Rabbit mAb #9582 (right). Note that Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb fails to detect poly-ubiquitinated β-catenin in MG132-treated cells, indicating its specificity for stabilized protein.
No image available
Immunohistochemistry Image 1: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin embedded human breast carcinoma using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb.
Immunohistochemistry Image 2: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded Apc (Min/+) mouse intestinal adenoma (left) and adjacent normal intestinal epithelium (right) using Non-phospho (Active) beta-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb. Note the nuclear accumulation of active beta-Catenin in the adenoma cells.
Immunohistochemistry Image 3: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin embedded cell pellets, HeLa (left) or NCI-H28 (right), using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb.
Immunohistochemistry Image 4: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin embedded mouse colon using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb in the presence of phospho-β-catenin (Ser33/37/Thr41) peptide (left) or non-phospho-β-catenin (Ser33/37/Thr41) peptide (right). Note the absence of staining only in the presence of the non-phospho-β-catenin (Ser33/37/Thr41) peptide, demonstrating non-phospho specificity.
Immunofluorescence Image 1: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HCT-15 (left, center; positive) or NCI-H28 (right; negative) cells using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb (green) in the presence of Phospho-Catenin-β (Ser33/Ser37/Thr41) peptide (left) or Non-phospho-Catenin-β (Ser33/Ser37/Thr41) peptide (center). Red = Propidium Iodide (PI)/RNase Staining Solution #4087.
Flow Cytometry Image 1: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
Flow cytometric analysis of K562 cells, untreated (blue) or treated with MG-132 #2194 (10 µM, 4 hr; green) using Non-phospho (Active) B-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E mAb IgG XP Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 cells and either TCF4 (C48H11) Rabbit mAb #2569 or Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. TCF4 and β-Catenin are known to associate with each other on chromatin. The figure shows binding of both TCF4 and β-Catenin across CAMK2D, a known target gene of both TCF4 and β-Catenin (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product datasheet.
Chromatin Immunoprecipitation Image 2: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 cells and either TCF4 (C48H11) Rabbit mAb #2569 or Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. TCF4 and β-Catenin are known to associate with each other on chromatin. The figure shows binding of both TCF4 and β-Catenin across chromosome 4 (upper), including CAMK2D (lower), a known target gene of both TCF4 and β-Catenin (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT 116 cells and either Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human Axin2 Intron 1 Primers #8973, SimpleChIP® Human CaMK2D Intron 3 Primers #5111, human c-Myc promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT and RUN Image 1: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
CUT&RUN was performed with HCT 116 cells and Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across Axin2, a known target gene of β-Catenin (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
CUT&RUN was performed with HCT 116 cells and Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 17 (upper), including Axin2 (lower), a known target gene of β-Catenin (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Expand Image
CUT&RUN was performed with HCT 116 cells and either Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human Axin2 Intron 1 Primers #8973, human c-Myc promoter primers, SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.