Revision 2

#68309Store at -20C

1 Kit

(8 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
Notch1 (D1E11) XP® Rabbit mAb 3608 20 µl 120, 300 kDa Rabbit IgG
Cleaved Notch1 (Val1744) (D3B8) Rabbit mAb 4147 20 µl 110 kDa Rabbit IgG
RBPSUH (D10A4) XP® Rabbit mAb 5313 20 µl 61 kDa Rabbit IgG
MAML1 (D3K7B) Rabbit mAb 12166 20 µl 130 kDa Rabbit IgG
c-Myc (D84C12) Rabbit mAb 5605 20 µl 57-65 kDa Rabbit IgG
p21 Waf1/Cip1 (12D1) Rabbit mAb 2947 20 µl 21 kDa Rabbit IgG
HES1 (D6P2U) Rabbit mAb 11988 20 µl 30 kDa Rabbit IgG
Cyclin D3 (DCS22) Mouse mAb 2936 20 µl 31 kDa Mouse IgG1
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl Horse 

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

Description

The Notch Activated Targets Antibody Sampler Kit provides an economical means of detecting target proteins of activated Notch. The kit contains enough primary antibody to perform four western blot experiments per primary 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

Notch proteins (Notch1-4) are a family of transmembrane receptors that play important roles in development and the determination of cell fate (1). Mature Notch receptors are processed and assembled as heterodimeric proteins, with each dimer comprised of a large extracellular ligand-binding domain, a single-pass transmembrane domain, and a smaller cytoplasmic subunit (Notch intracellular domain, NICD) (2). Binding of Notch receptors to ligands of the Delta-Serrate-Lag2 (DSL) family triggers heterodimer dissociation, exposing the receptors to proteolytic cleavages; these result in release of the NICD, which translocates to the nucleus and activates transcription of downstream target genes (3,4).  RBPSUH (Recombining Binding Protein, SUppressor of Hairless), is the DNA-binding component of the transcription complex regulated by canonical Notch signaling. Binding of Notch with RBPSUH activates a transcription activation complex that includes Mastermind-like (MAML) proteins, leading to transcriptional activation of Notch target genes (5-7). The NICD binds and activates c-Myc which functions as a transcriptional regulator with roles in various aspects of cell behavior including proliferation, differentiation and apoptosis (8).  The tumor suppressor protein p21 Waf1/Cip1 acts as an inhibitor of cell cycle progression. The NICD-RBPSUH complex binds and activates p21 for transcription (15).  HES1 (Hairy and Enhancer of Split 1) is one of seven members of the HES family of basic helix-loop-helix (bHLH) transcription factors that is particularly well known as a repressive mediator of the canonical Notch signaling pathway (10). HES1 plays a key role in mediating Notch-dependent T cell lineage commitment (11), and has been reported to be an essential mediator of Notch-induced T cell acute lymphoblastic leukemia (T-ALL) (11,12). The active complex of cyclin D/CDK4 targets the retinoblastoma protein for phosphorylation, allowing the release of E2F transcription factors that activate G1/S-phase gene expression (13).  Transcription of cyclin D is in part regulated by the NICD binding to the promoter region of cyclin D (14).

  1. Artavanis-Tsakonas, S. et al. (1999) Science 284, 770-6.
  2. Chan, Y.M. and Jan, Y.N. (1998) Cell 94, 423-6.
  3. Schroeter, E.H. et al. (1998) Nature 393, 382-6.
  4. Rand, M.D. et al. (2000) Mol Cell Biol 20, 1825-35.
  5. Wu, L. et al. (2002) Mol Cell Biol 22, 7688-700.
  6. Lin, S.E. et al. (2002) J Biol Chem 277, 50612-20.
  7. Kitagawa, M. et al. (2001) Mol Cell Biol 21, 4337-46.
  8. Baudino, T.A. and Cleveland, J.L. (2001) Mol Cell Biol 21, 691-702.
  9. Flores-Rozas, H. et al. (1994) Proc Natl Acad Sci U S A 91, 8655-9.
  10. Kobayashi, T. and Kageyama, R. (2010) Genes Cells 15, 689-98.
  11. Wendorff, A.A. et al. (2010) Immunity 33, 671-84.
  12. Espinosa, L. et al. (2010) Cancer Cell 18, 268-81.
  13. Lukas, J. et al. (1996) Mol Cell Biol 16, 6917-25.
  14. Li, X. and von Boehmer, H. (2011) ISRN Hematol 2011, 921706.
  15. Niimi, H. et al. (2007) J Cell Biol 176, 695-707.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP 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

    法的な権限を与えられたCSTの担当者が署名した書面によって別途明示的に合意された場合を除き、 CST、その関連会社または代理店が提供する製品には以下の条件が適用されます。お客様が定める条件でここに定められた条件に含まれるものを超えるもの、 または、ここに定められた条件と異なるものは、法的な権限を与えられたCSTの担当者が別途書面にて受諾した場合を除き、拒絶され、 いかなる効力も効果も有しません。

    研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。

    Revision 2
    #68309

    Notch Activated Targets Antibody Sampler Kit

    Notch Activated Targets Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (1.0 mg/mL) from MCF-7 cells using p21 Waf1/Cip1 (12D1) Rabbit mAb #2947. The virtual lane view (left) shows a single 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.
    Notch Activated Targets Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from Raji cells using c-Myc (D84C12) Rabbit mAb #5605. The virtual lane view (left) shows a single target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (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.
    Notch Activated Targets Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from various cell lines using HES1 (D6P2U) Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using MAML1 (D3K7B) Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from SK-N-MC, C6 and IMCD3 cells, using Cyclin D3 (DCS22) Mouse mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis from control HeLa cells (lane 1) or p21 Waf1/Cip1 knockout HeLa cells (lane 2) using p21 Waf1/Cip1 (12D1) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the p21 Waf1/Cip1 knockout HeLa cells confirms specificity of the antibody for p21 Waf1/Cip1.
    Notch Activated Targets Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of total cell extract from various cell types using Notch1 (D1E11) XP® Rabbit mAb. The full-length (FL) Notch protein and the cleaved transmembrane/intracellular region (NTM) are indicated.
    Notch Activated Targets Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from various cell lines using Cleaved Notch1 (Val1744) (D3B8) Rabbit mAb (upper) or Notch1 (D1E11) XP® Rabbit mAb #3608 (lower).
    Notch Activated Targets Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from various cell lines using RBPSUH (D10A4) XP® Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from control HEK293 cells (lane 1) or c-Myc knockout HEK293 cells (lane 2) using c-Myc (D84C12) Rabbit mAb Antibody, #5605 (upper) or β-actin (13E5) Rabbit mAb, #4970 (lower). The absence of signal in the c-Myc knockout HEK293 cells confirms specificity of the antibody for c-Myc.
    Notch Activated Targets Antibody Sampler Kit: Image 11 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.
    Notch Activated Targets Antibody Sampler Kit: Image 12 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.
    Notch Activated Targets Antibody Sampler Kit: Image 13 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using HES1 (D6P2U) Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 14 Expand Image
    Immunoprecipitation of MAML1 protein from 293T cell extracts, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or MAML1 (D3K7B) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using MAML1 Antibody #4608.
    Notch Activated Targets Antibody Sampler Kit: Image 15 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Cyclin D3 (DCS22) Mouse mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 16 Expand Image
    Western blot analysis of extracts from various cell types using p21 Waf1/Cip1 (12D1) Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 17 Expand Image
    Immunohistochemical analysis of paraffin-embedded human inflammatory granulation tissue using Notch1 (D1E11) XP® Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 18 Expand Image
    CUTLL1 cells were cultured in media with γ-secretase inhibitor (1μM) for 3 days and then either harvested immediately (left panel) or washed and cultured in fresh media for 3h (right panel). Chromatin immunoprecipitations were performed with cross-linked chromatin from cells and Cleaved Notch1 (Val1744) (D3B8) 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 human HES1 promoter primers, SimpleChIP® Human HES4 Promoter Primers #7273, 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.
    Notch Activated Targets Antibody Sampler Kit: Image 19 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using RBPSUH (D10A4) XP® Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 20 Expand Image
    Western blot analysis of extracts from HeLa cells, mock transfected or transfected with SignalSilence® c-Myc siRNA I #6341, using c-Myc (D84C12) Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 21 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using HES1 (D6P2U) Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 22 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from CUTLL1 cells cultured in media with γ-secretase inhibitor (1 μM) for 3 days and then washed and cultured in fresh media for 3 hr and either MAML1 (D3E9) Rabbit mAb #11959 or MAML1 (D3K7B) Rabbit mAb, using SimpleChIP® Plus 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 DTX1, a known target gene of MAML1 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product datasheet.
    Notch Activated Targets Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Cyclin D3 (DCS22) Mouse mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 24 Expand Image
    Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® p21 Waf1/Cip1 siRNA II (+), using p21 Waf1/Cip1 (12D1) Rabbit mAb #2947 and α-Tubulin (11H10) Rabbit mAb #2125. The p21 Waf1/Cip1 (12D1) Rabbit mAb confirms silencing of p21 Waf1/Cip1 expression and α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of p21 Waf1/Cip1 siRNA.
    Notch Activated Targets Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Notch1 (D1E11) XP® Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 26 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using RBPSUH (D10A4) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    Notch Activated Targets Antibody Sampler Kit: Image 27 Expand Image
    Western blot analysis of extracts from various cell lines using c-Myc (D84C12) Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded CUTLL1 cell pellets, control (left) or Compound E-treated (right), using HES1 (D6P2U) Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 29 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from CUTLL1 cells cultured in media with γ-secretase inhibitor (1 μM) for 3 days and then washed and cultured in fresh media for 3 hr and either MAML1 (D3E9) Rabbit mAb #11959 or MAML1 (D3K7B) Rabbit mAb, using SimpleChIP® Plus 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 12 (upper), including DTX1 (lower), a known target gene of MAML1 (see additional figure containing ChIP-qPCR data).
    Notch Activated Targets Antibody Sampler Kit: Image 30 Expand Image
    Immunoprecipitation of p21 from human umbillical vein endothelial cells (HUVECs) using p21 Waf1/Cip1 (12D1) Rabbit mAb. Western blot detection was performed using the same antibody.
    Notch Activated Targets Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded A2780 (left), Jurkat (center) and RL (right) cell pellets using Notch1 (D1E11) XP® Rabbit mAb. Both A2780 and Jurkat express Notch1, but only Jurkat cells have cleaved Notch1, while RL cells express very low or no Notch1.
    Notch Activated Targets Antibody Sampler Kit: Image 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded E18.5 mouse lung, Rbpjk F/+ Shh+/+ (wild type, left) or Rbpjk F/- Shhcre/+ (Rbpjk conditional knock out, right), using RBPSUH (D10A4) XP® Rabbit mAb. Note lack of staining in the bronchial epithelial cells in the conditional knock out tissue (right). Tissue courtesy of Dr. Wellington Cardosa, Boston University School of Medicine.
    Notch Activated Targets Antibody Sampler Kit: Image 33 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, mock-transfected (left) or transfected with SignalSilence® c-Myc siRNA I #6341 (right), using c-Myc (D84C12) Rabbit mAb (green). Actin filaments have been labeled wth DY-554 phalloidin (red).
    Notch Activated Targets Antibody Sampler Kit: Image 34 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from CUTLL1 cells cultured in media with γ-secretase inhibitor (1 μM) for 3 days and then washed and cultured in fresh media for 3 hr and either MAML1 (D3K7B) 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 human DTX1 intron 3 primers, SimpleChIP® Human HES4 Promoter Primers #7273, 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.
    Notch Activated Targets Antibody Sampler Kit: Image 35 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using p21 Waf1/Cip1 (12D1) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    Notch Activated Targets Antibody Sampler Kit: Image 36 Expand Image
    Immunohistochemical analysis of paraffin-embedded human stomach adjacent to MALT (mucosa-associated lymphoid tissue) lymphoma using Notch1 (D1E11) XP® Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 37 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using RBPSUH (D10A4) XP® Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 38 Expand Image
    Immunohistochemical analysis of paraffin-embedded HeLa cells, transfected with SignalSilence® Control siRNA (Unconjugated) #6568 (left) or SignalSilence® p21 Waf1/Cip1 siRNA II #6558 (right), using p21 Waf1/Cip1 (12D1) Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 39 Expand Image
    CUTLL1 cells were cultured in media with γ-secretase inhibitor (1 μM) for 3 days and then either harvested immediately (left panel) or washed and cultured in fresh media for 3 hr (right panel). Chromatin immunoprecipitations were performed with cross-linked chromatin from cells and Notch1 (D1E11) 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 human HES1 promoter primers, SimpleChIP® Human HES4 Promoter Primers #7273, 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.
    Notch Activated Targets Antibody Sampler Kit: Image 40 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse lymph node using RBPSUH (D10A4) XP® Rabbit mAb.
    Notch Activated Targets Antibody Sampler Kit: Image 41 Expand Image
    Confocal immunofluorescent analysis of MCF7 cells using p21 Waf1/Cip1 (12D1) Rabbit mAb (red) and Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Notch Activated Targets Antibody Sampler Kit: Image 42 Expand Image
    CUTLL1 cells were cultured in media with γ-secretase inhibitor (1 μM, 3 d) and then either harvested immediately (left panel) or washed and cultured in fresh media for 3 h (right panel). Chromatin immunoprecipitations were performed with cross-linked chromatin from cells and RBPSUH (D10A4) 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 human HES1 promoter primers, SimpleChIP® Human HES4 Promoter Primers #7273, 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.
    Notch Activated Targets Antibody Sampler Kit: Image 43 Expand Image
    Flow cytometric analysis of Daudi cells using p21 Waf1/Cip1 (12D1) Rabbit mAb (right) and Propidium Iodide (PI)/RNase Staining Solution #4087, compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.