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® Cleaved Notch1 (Val1744) Sandwich ELISA Kit #7194 is shown.
Figure 1. Jurkat cells express cleaved Notch1 (Val1744) as detected by the PathScan® Cleaved Notch1 (Val1744) Sandwich ELISA Kit #7194. Treatment with 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.
|Product Includes||Volume||Solution Color|
|Notch1 Rat mAb Coated Microwells||96 tests|
|Cleaved Notch1 (Val1744) Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml|
|Cell Lysis Buffer (10X) 9803||15 ml|
PathScan® Cleaved Notch1 (Val1744) Sandwich ELISA Kit from Cell Signaling Technology is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Notch1 when cleaved at Val1744. 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 cleaved Notch1 (Val1744) Rabbit detection 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 cleaved Notch1 (Val1744).
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Cleaved Notch1 (Val1744) Sandwich ELISA Kit #7194 detects endogenous levels of Notch1 protein when cleaved at Val1744 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.
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).
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