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68309
Notch Activated Targets Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit
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Notch Activated Targets Antibody Sampler Kit #68309

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Notch Activated Targets Antibody Sampler Kit: Image 1
Western blot analysis of extracts from various cell lines using HES1 (D6P2U) Rabbit mAb.
Notch Activated Targets Antibody Sampler Kit: Image 2
Western blot analysis of extracts from various cell lines using MAML1 (D3K7B) Rabbit mAb.
Notch Activated Targets Antibody Sampler Kit: Image 3
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 4
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.
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Notch Activated Targets Antibody Sampler Kit: Image 5
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 6
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 7
Western blot analysis of extracts from various cell lines using RBPSUH (D10A4) XP® Rabbit mAb.
Notch Activated Targets Antibody Sampler Kit: Image 8
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.
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Notch Activated Targets Antibody Sampler Kit: Image 9
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 10
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 11
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using HES1 (D6P2U) Rabbit mAb.
Notch Activated Targets Antibody Sampler Kit: Image 12
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 13
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Cyclin D3 (DCS22) Mouse mAb.
Notch Activated Targets Antibody Sampler Kit: Image 14
Western blot analysis of extracts from various cell types using p21 Waf1/Cip1 (12D1) Rabbit mAb.
Notch Activated Targets Antibody Sampler Kit: Image 15
Immunohistochemical analysis of paraffin-embedded human inflammatory granulation tissue using Notch1 (D1E11) XP® Rabbit mAb.
Notch Activated Targets Antibody Sampler Kit: Image 16
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.
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Notch Activated Targets Antibody Sampler Kit: Image 17
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using RBPSUH (D10A4) XP® Rabbit mAb.
Notch Activated Targets Antibody Sampler Kit: Image 18
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 19
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using HES1 (D6P2U) Rabbit mAb.
Notch Activated Targets Antibody Sampler Kit: Image 20
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 data sheet.
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Notch Activated Targets Antibody Sampler Kit: Image 21
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Cyclin D3 (DCS22) Mouse mAb.
Notch Activated Targets Antibody Sampler Kit: Image 22
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.
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Notch Activated Targets Antibody Sampler Kit: Image 23
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Notch1 (D1E11) XP® Rabbit mAb.
Notch Activated Targets Antibody Sampler Kit: Image 24
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 25
Western blot analysis of extracts from various cell lines using c-Myc (D84C12) Rabbit mAb.
Notch Activated Targets Antibody Sampler Kit: Image 26
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 27
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).
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Notch Activated Targets Antibody Sampler Kit: Image 28
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 29
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 30
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.
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Notch Activated Targets Antibody Sampler Kit: Image 31
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 32
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.
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Notch Activated Targets Antibody Sampler Kit: Image 33
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 34
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 35
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using RBPSUH (D10A4) XP® Rabbit mAb.
Notch Activated Targets Antibody Sampler Kit: Image 36
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 37
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.
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Notch Activated Targets Antibody Sampler Kit: Image 38
Immunohistochemical analysis of paraffin-embedded mouse lymph node using RBPSUH (D10A4) XP® Rabbit mAb.
Notch Activated Targets Antibody Sampler Kit: Image 39
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 40
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.
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Notch Activated Targets Antibody Sampler Kit: Image 41
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.
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To Purchase # 68309
Cat. # Size Qty. Price
68309T		 1 Kit  (8 x 20 microliters)
$ 592

Custom Formulation

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Notch1 (D1E11) XP® Rabbit mAb 3608 20 µl
  • WB
  • IP
  • IHC
  • ChIP
 H M R 120, 300 Rabbit IgG
Cleaved Notch1 (Val1744) (D3B8) Rabbit mAb 4147 20 µl
  • WB
  • IP
  • ChIP
 H M R 110 Rabbit IgG
RBPSUH (D10A4) XP® Rabbit mAb 5313 20 µl
  • WB
  • IHC
  • ChIP
 H M R Mk 61 Rabbit IgG
MAML1 (D3K7B) Rabbit mAb 12166 20 µl
  • WB
  • IP
  • ChIP
 H M R 130 Rabbit IgG
c-Myc (D84C12) Rabbit mAb 5605 20 µl
  • WB
  • IF
 H M R 57-65 Rabbit IgG
p21 Waf1/Cip1 (12D1) Rabbit mAb 2947 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
 H Mk 21 Rabbit IgG
HES1 (D6P2U) Rabbit mAb 11988 20 µl
  • WB
  • IP
  • IHC
 H M R Mk 30 Rabbit IgG
Cyclin D3 (DCS22) Mouse mAb 2936 20 µl
  • WB
  • IHC
 H M R 31 Mouse IgG1
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
 Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
 Horse 

Product 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.

Specificity / Sensitivity

Notch1 (D1E11) XP® Rabbit mAb detects intracellular epitopes between 2400 and 2500 amino acids of human Notch1. It recognizes both the full-length (~300 kDa) and the NTM region (~120 kDa). The antibody cannot detect the extracellular (ligand-binding) domain of Notch1 following cleavage at the S2 site by ADAM-type metalloproteases. Cleaved Notch1 (V1744) (D3B8) Rabbit mAb detects endogenous levels of the Notch1 intracellular domain (NICD) only when released by cleavage between Gly1753 and Val1754 (equivalent to Gly1743/Val1744 of murine notch1). The antibody does not recognize full-length Notch1 or Notch1 cleaved at other positions. The size of the NICD varies among cell lines due to mutations in the Notch1 C-terminus. RBPSUH (D10A4) XP® Rabbit mAb recognizes endogenous levels of total RBPSUH protein. MAML1 (D3K7B) Rabbit mAb recognizes endogenous levels of total MAML1 protein. This antibody does not detect MAML2 or MAML3. c-Myc (D84C12) Rabbit mAb recognizes endogenous levels of total c-Myc protein. p21 Waf1/Cip1 (12D1) Rabbit mAb detects endogenous levels of total p21 protein. The antibody does not cross-react with other CDK inhibitors. HES1 (D6P2U) Rabbit mAb recognizes endogenous levels of total HES1 protein. Cyclin D3 (DCS22) Mouse mAb detects endogenous levels of total cyclin D3 protein. The antibody does not cross-react with cyclin D1 or cyclin D2. 

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro2438 of human Notch1, the Val1754 cleavage site in human Notch1 (equivalent to Val1744 in mouse Notch1), residues surrounding Gln110 of human RBPSUH protein, residues surrounding Asp269 of human MAML1 protein, amino-terminal residues of c-Myc, residues near the carboxy-terminus of human p21, recombinant protein specific to human HES1 protein, residues 241-260 of recombinant human cyclin D3. Antibodies are purified by protein A and peptide affinity chromatography.

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.

Pathways & Proteins

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Limited Uses

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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.

For Research Use Only. Not For Use In Diagnostic Procedures.
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