Revision 1

#2951Store at -20C

1 Kit

(5 x 20 microliters)

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.
Product Includes Product # Quantity Mol. Wt Isotype/Source
β-Catenin (D10A8) XP® Rabbit mAb 8480 20 µl 92 kDa Rabbit IgG
Phospho-β-Catenin (Ser33/37/Thr41) Antibody 9561 20 µl 92 kDa Rabbit 
Phospho-β-Catenin (Thr41/Ser45) Antibody 9565 20 µl 92 kDa Rabbit 
Phospho-β-Catenin (Ser552) (D8E11) Rabbit mAb 5651 20 µl 92 kDa Rabbit IgG
Phospho-β-Catenin (Ser675) (D2F1) XP® Rabbit mAb 4176 20 µl 92 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The β-Catenin Antibody Sampler Kit provides an economical means of detecting total β-catenin as well as β-catenin phosphorlylated at various residues. The kit contains enough primary and secondary antibody to perform two Western blots with each antibody.

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.

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).

Both Akt and PKA were shown to phosphorylate β-catenin at Ser552 and Ser675. Phosphorylation at Ser552 and Ser675 induces β-catenin accumulation in the nucleus and increases its transcriptional activity (9-12).

  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.
  9. Taurin, S. et al. (2006) J. Biol. Chem. 281, 9971-9976.
  10. Fang, D. et al. (2007) J. Biol. Chem. 282, 11221-11229.
  11. He, X.C. et al. (2007) Nat. Genet. 39, 189-198.
  12. Hino, S. et al. (2005) Mol. Cell Biol. 25, 9063-9072.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #2951

    β-Catenin Antibody Sampler Kit

    β-Catenin Antibody Sampler Kit: Image 1 Expand Image
    Immunoprecipitation of β-Catenin from HeLa cell extracts. Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is β-Catenin (D10A8) XP® Rabbit mAb, #8480. Western blot was performed using β-Catenin (15B8) Mouse mAb, #37477.
    β-Catenin Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from HeLa cells using β-Catenin (D10A8) XP® Rabbit mAb #8480. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    β-Catenin Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from control HeLa cells (lane 1) or HeLa cells with a targeted mutation in the gene encoding β-Catenin (lane 2) using Phospho-β-Catenin (Ser675) (D2F1) XP® Rabbit mAb (upper), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The change in β-Catenin molecular weight in the mutated HeLa cells confirms the specificity of the antibody for β-Catenin.
    β-Catenin Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from SK-N-MC and NTERA-2 cells, untreated or λ phosphatase-treated, using

    Phospho-β-Catenin (Ser552) (D8E11) Rabbit mAb (upper) or β-Catenin (6B3) Rabbit mAb #9582 (lower).

    β-Catenin Antibody Sampler Kit: Image 5 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    β-Catenin Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from control HeLa cells (lane 1) or HeLa cells with an apparent in-frame truncation mutation in the gene encoding β-Catenin (lane 2) using β-Catenin (D10A8) XP® Rabbit mAb, #8480 (upper) or β-actin (D6A8) Rabbit mAb #8457 (lower). The change in β-Catenin molecular weight in the mutated HeLa cells is consistent with an in-frame deletion.
    β-Catenin Antibody Sampler Kit: Image 7 Expand Image
    CUT&RUN was performed with HCT 116 cells and β-Catenin (D10A8) XP® 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).
    β-Catenin Antibody Sampler Kit: Image 8 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using ß-Catenin (D10A8) XP® Rabbit mAb performed on the Leica® BOND™ Rx.
    β-Catenin Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from 293 cells, pretreated with 20 mM LiCl for 30 minutes and then with 50 nM calyculin A, using Phospho-β-Catenin (Ser33/37/Thr41) Antibody (upper) or β-Catenin Antibody #9562 (lower).
    β-Catenin Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from 293 cells pretreated with 20 mM LiCl for 30 minutes and then with 50 nM calyculin A, using Phospho-β-Catenin (Thr41/Ser45) Antibody (upper) or β-Catenin Antibody #9562 (lower).
    β-Catenin Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from MKN-45 and SK-N-MC cells, untreated or treated with λ phosphatase for 1 hour or forskolin (FSK) for 30 minutes, using Phospho-β-Catenin (Ser675) (D2F1) XP® Rabbit mAb (upper) or β-Catenin (6B3) Rabbit mAb #9582 (lower).
    β-Catenin Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from various cell lines using β-Catenin (D10A8) XP® Rabbit mAb.
    β-Catenin Antibody Sampler Kit: Image 13 Expand Image
    CUT&RUN was performed with HCT 116 cells and β-Catenin (D10A8) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were 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).
    β-Catenin Antibody Sampler Kit: Image 14 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using ß-Catenin (D10A8) XP® Rabbit mAb performed on the Leica® BOND™ Rx.
    β-Catenin Antibody Sampler Kit: Image 15 Expand Image
    Western blot analysis of extracts from SW480 cells using Phospho-β-Catenin (Ser33/37/Thr41) Antibody.
    β-Catenin Antibody Sampler Kit: Image 16 Expand Image
    Western blot analysis of extracts from SW480 cells using Phospho-β-Catenin (Thr41/Ser45) Antibody.
    β-Catenin Antibody Sampler Kit: Image 17 Expand Image
    Confocal immunofluorescent analysis of rat colon using Phospho-β-Catenin (Ser675) (D2F1) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 Phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    β-Catenin Antibody Sampler Kit: Image 18 Expand Image
    CUT&RUN was performed with HCT 116 cells and either β-Catenin (D10A8) XP® 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, SimpleChIP® Human CaMK2D Intron 3 Primers #5111 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.
    β-Catenin Antibody Sampler Kit: Image 19 Expand Image
    Immunohistochemical analysis of paraffin-embedded human serous adenocarcinoma of the ovary using ß-Catenin (D10A8) XP® Rabbit mAb performed on the Leica® BOND™ Rx.
    β-Catenin Antibody Sampler Kit: Image 20 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, untreated (left), λ phosphatase-treated (middle), or untreated NCI-H28 cells (β-catenin null; right) using Phospho-β-Catenin (Ser675) (D2F1) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    β-Catenin Antibody Sampler Kit: Image 21 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using ß-Catenin (D10A8) XP® Rabbit mAb.

    β-Catenin Antibody Sampler Kit: Image 22 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using β-Catenin (D10A8) XP® Rabbit mAb.
    β-Catenin Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using ß-Catenin (D10A8) XP® Rabbit mAb.
    β-Catenin Antibody Sampler Kit: Image 24 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using β-Catenin (D10A8) XP® Rabbit mAb.
    β-Catenin Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using β-Catenin (D10A8) XP® Rabbit mAb.
    β-Catenin Antibody Sampler Kit: Image 26 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse colon using β-Catenin (D10A8) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    β-Catenin Antibody Sampler Kit: Image 27 Expand Image
    Immunohistochemical analysis of paraffin-embedded cell pellets, HeLa (left) or NCI-H28 (right), using β-Catenin (D10A8) XP® Rabbit mAb.
    β-Catenin Antibody Sampler Kit: Image 28 Expand Image
    Confocal immunofluorescent analysis of mouse colon using β-Catenin (D10A8) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    β-Catenin Antibody Sampler Kit: Image 29 Expand Image
    Confocal immunofluorescent analysis of HeLa (left) and NCI-H28 (right) cells using β-Catenin (D10A8) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    β-Catenin Antibody Sampler Kit: Image 30 Expand Image
    Flow cytometric analysis of NCI-H28 cells (green) and HeLa cells (blue) using β-Catenin (D10A8) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as secondary antibody.
    β-Catenin Antibody Sampler Kit: Image 31 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 cells and either β-Catenin (D10A8) XP® Rabbit mAb or Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb #8814, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were 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 ChIP-qPCR data).
    β-Catenin Antibody Sampler Kit: Image 32 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 cells and either β-Catenin (D10A8) XP® Rabbit mAb or Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb #8814, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 17 (upper), including AXIN2 (lower), a known target gene of β-Catenin (see additional figure containing ChIP-qPCR data).
    β-Catenin Antibody Sampler Kit: Image 33 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT 116 cells and either β-Catenin (D10A8) XP® 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.
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.