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3640
Notch Isoform Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Notch Isoform Antibody Sampler Kit #3640

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Notch Isoform Antibody Sampler Kit: Image 1

Western blot analysis of extracts from 293 and SK-MEL-5 cells using Notch2 (D76A6) XP® Rabbit mAb #5732. The full-length (FL) Notch2 protein and the transmembrane/intracellular region (NTM) are indicated.

Notch Isoform Antibody Sampler Kit: Image 2

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 Isoform Antibody Sampler Kit: Image 3

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 Isoform Antibody Sampler Kit: Image 4

Western blot analysis of extracts from various cell lines using Notch3 (D11B8) Rabbit mAb.

Notch Isoform Antibody Sampler Kit: Image 5

Flow cytometric analysis of fixed and permeabilized SW620 cells (red, low expression) and 293 cells (blue, positive) cells using Notch2 (D76A6) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Notch Isoform Antibody Sampler Kit: Image 6

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 Isoform Antibody Sampler Kit: Image 7

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 Isoform Antibody Sampler Kit: Image 8

Immunohistochemical analysis of paraffin-embedded human inflammatory granulation tissue using Notch1 (D1E11) XP® Rabbit mAb.

Notch Isoform Antibody Sampler Kit: Image 9

Confocal immunofluorescent analysis of SNB19 cells using Notch2 (D76A6) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Notch Isoform Antibody Sampler Kit: Image 10

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Notch1 (D1E11) XP® Rabbit mAb.

Notch Isoform Antibody Sampler Kit: Image 11

Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma (left) or endometrioid adenocarcinoma (right) using Notch2 (D76A6) XP® Rabbit mAb (top) or Notch2 Rabbit mAb (bottom). These two antibodies detect independent, unique epitopes on human Notch2. Both epitopes are contained within the intracellular domain and the nuclear staining represents active Notch2. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.

Notch Isoform Antibody Sampler Kit: Image 12

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 Isoform Antibody Sampler Kit: Image 13

Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using Notch2 (D76A6) XP® Rabbit mAb.

Notch Isoform Antibody Sampler Kit: Image 14

Immunohistochemical analysis of paraffin-embedded human stomach adjacent to MALT (mucosa-associated lymphoid tissue) lymphoma using Notch1 (D1E11) XP® Rabbit mAb.

Notch Isoform Antibody Sampler Kit: Image 15

Immunohistochemical analysis of paraffin-embedded 293T cell pellet (left, positive) or IMR-32 cell pellet (right, negative) using Notch2 (D76A6) XP® Rabbit mAb.

Notch Isoform Antibody Sampler Kit: Image 16

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 Isoform Antibody Sampler Kit: Image 17

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Notch2 (D76A6) XP® Rabbit mAb.

Notch Isoform Antibody Sampler Kit: Image 18

Immunohistochemical analysis of paraffin-embedded human B-cell non-Hodgkin's lymphoma using Notch2 (D76A6) XP® Rabbit mAb.

Notch Isoform Antibody Sampler Kit: Image 19

Immunohistochemical analysis of paraffin-embedded human ovarian serous carcinoma using Notch2 (D76A6) XP® Rabbit mAb.

Notch Isoform Antibody Sampler Kit: Image 20

Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using Notch2 (D76A6) XP® Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).

Notch Isoform Antibody Sampler Kit: Image 21

Immunohistochemical analysis of paraffin-embedded human placenta using Notch2 (D76A6) XP® Rabbit mAb.

Notch Isoform Antibody Sampler Kit: Image 22

Immunohistochemical analysis of paraffin-embedded human testis using Notch2 (D76A6) XP® Rabbit mAb.

Notch Isoform Antibody Sampler Kit: Image 23

Western blot analysis of extracts from 293 and SK-MEL-5 cells using Notch2 (D76A6) XP® Rabbit mAb. The full-length (FL) Notch2 protein and the transmembrane/intracellular region (NTM) are indicated.

To Purchase # 3640T
Product # Size Price
3640T
1 Kit  (4 x 20 µl) $ 327

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Cleaved Notch1 (Val1744) (D3B8) Rabbit mAb 4147 20 µl
  • WB
  • IP
  • ChIP
H M R 110 Rabbit IgG
Notch1 (D1E11) XP® Rabbit mAb 3608 20 µl
  • WB
  • IP
  • IHC
  • ChIP
H M R 120, 300 Rabbit IgG
Notch3 (D11B8) Rabbit mAb 5276 20 µl
  • WB
  • IP
H 90, 270 Rabbit IgG
Notch2 (D76A6) XP® Rabbit mAb 5732 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R 110, 300 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The Notch Isoform Antibody Sampler Kit provides an economical means to investigate Notch Signaling. The kit contains primary and secondary antibodies to perform two western mini-blots with each antibody.

Specificity / Sensitivity

Cleaved Notch1 (V1744) (D3B8) Rabbit mAb detects endogenous levels of the Notch1 intra- cellular 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 (6). Notch1 (D1E11) XP® Rabbit mAb detects endogenous levels of total Notch1 protein. It recognizes both the full-length (~300 kDa) and the trans- membrane/intracellular region NTM (~120 kDa). Notch2 (D76A6) XP® Rabbit mAb detects endogenous levels of total Notch2 protein. It recognizes both the full-length (~ 300 kDa) and the trans- membrane/intracellular region NTM (~110 kDa). Notch3 (D11B8) Rabbit mAb detects endogenous levels of total Notch3 protein. The antibody recognizes both full-length (FL) Notch3 at 270 kDa and a truncated protein (NTM) containing a short extracellullar region, the transmembrane domain and the intracellular region at 90 kDa.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence at the Val1754 cleavage site in human Notch1 (equivalent to Val1744 in mouse Notch1), Pro2438 of human Notch1, Ala2738 of human Notch2, or Glu2312 of human Notch3 protein.

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).

Constitutively activated Notch1 signaling is associated with the majority of cases of T cell acute lymphoblastic leukemia (T-ALL). The activation is either due to mutations in Notch1 itself or in the components of ubiquitin ligase complex, namely FBW7 (5-6). Notch2 is a member of the Notch family and mutation in Notch2 is associated with Alagille syndrome (7). Notch3 is a member of the Notch family and is processed similar to Notch1 (8). It is expressed primarily in arterial smooth muscle cells (SMC). Mutations altering the number of cysteine residues in the notch3 extracellular region are associated with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary angiopathy leading to strokes and dementia in adults (9-11). Recent studies indicate that Notch3 is overexpressed in many types of cancer (12-14).

  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. Weng, A.P. et al. (2004) Science 306, 269-71.
  6. Thompson, B.J. et al. (2007) J Exp Med 204, 1825-35.
  7. McDaniell, R. et al. (2006) Am J Hum Genet 79, 169-73.
  8. Baron, M. (2003) Semin Cell Dev Biol 14, 113-9.
  9. Kalimo, H. et al. (2002) Brain Pathol 12, 371-84.
  10. Karlström, H. et al. (2002) Proc Natl Acad Sci U S A 99, 17119-24.
  11. Monet, M. et al. (2007) Hum Mol Genet 16, 982-92.
  12. Park, J.T. et al. (2006) Cancer Res 66, 6312-8.
  13. Gramantieri, L. et al. (2007) Liver Int 27, 997-1007.
  14. Yamaguchi, N. et al. (2008) Cancer Res 68, 1881-8.

Limited Uses

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