Western blot analysis of extracts from HBP-ALL and A204 cells using Notch3 (8G5) Rat mAb. Full-length (FL) and cleaved notch3 proteins are indicated.
|MW (kDa)||90, 270|
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.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 340
Notch3 (8G5) Rat mAb detects endogenous levels of total notch3 protein. It recognizes the full-length (270 kDa) and the extracellular truncated fragment containing a short extracellular region, the transmembrane domain and the intracellular region (90 kDa).
Monoclonal antibody is produced by immunizing animals with a fusion protein corresponding to intracellular residues of notch3.
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).
Notch3 is a member of notch family that is processed in a similar way to notch1 (5). 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 (6-8). Recent studies indicate that notch3 is overexpressed in many types of cancers (9-11).
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