Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total Notch1 Sandwich ELISA Kit #7245

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Kit Includes Volume Solution Color
Notch1 Rat Antibody Coated Microwells 96 tests
Notch1 Rabbit Detection Ab 11 ml Green
Anti-rabbit IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

PathScan® Total Notch1 Sandwich ELISA Kit from Cell Signaling Technology is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Notch1. A Notch1 rat antibody has been coated onto the microwells. After incubation with cell lysates, Notch1 protein is captured by the coated antibody. Following extensive washing, a Notch1 rabbit antibody is added to detect the captured Notch1 protein. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Notch1.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Total Notch1 Sandwich ELISA Kit #7245 detects endogenous levels of Notch1 protein as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA

ELISA

Figure 2. The relationship between the protein concentration of lysates from Jurkat cells, untreated or treated with γ secretase inhibitor, and the absorbance at 450 nm using the PathScan® Total Notch1 Sandwich ELISA Kit #7245 is shown.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Both cleaved and noncleaved Notch1 can be detected by the PathScan® Total Notch1 Sandwich ELISA Kit #7245. Jurkat cells express cleaved Notch1 (Val1744) and γ secretase inhibitor inhibits the cleavage of Notch1. Jurkat cells were treated with 25 μM γ secretase inhibitor for 96 hours. The absorbance readings at 450 nm are shown on the top, while the corresponding western blots using Notch1 (D1E11) XP® Rabbit mAb #3608 (left panel) or Cleaved Notch1 (Val1744) Antibody #2421 (right panel) are shown on the bottom.

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).

  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.

Application References

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Protocols

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

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