Anti-Notch1, intracellular domain (Notch Homolog Protein 1, hN1, Translocation-associated Notch Prot

Notch-1 (so named for "notches" in fly wings, also TAN-1) is a 300kD member of the Notch family of glycoproteins. It is associated with gene activation in both embryo and adult. Human Notch-1 is a 2538 amino acid (aa) type I transmembrane glycoprotein. It undergoes Golgi processing to generate a heterodimer composed of a 180-200kD disulfide-linked , extracellular domain (aa 18-1664) and a 120kD membrane- bound segment aa 1665-2556). Upon ligand binding, the 110kD segment undergoes two cleavages which generate an NICD (notch intracellular domain) (aa 1754-2556), a nuclear transcription factor. One isoform shows a deletion of aa 248-288. Applications: Suitable for use in for use in Western blot, Immunocytochemistry, Flow cytometry and Direct ELISA. Other applications have not been tested. , , Recommended Dilution: , Western Blot: 0.1-0.2 ug/ml. The detection limit for rhNotch-1 is ~ 0.5 ng/lane under non-reducing and reducing conditions. , Immunocytochemistry: 10ug/ml to detect human Notch-1 in SAOS human osteosarcoma cells. Cells were fixed with PBS containing 4% paraformaldehyde for 20 minutes at RT and blocked with PBS containing 10% normal donkey serum, 0.1% Triton X-100, and 1% BSA for 45 minutes at RT. After blocking, cells were incubated with diluted primary antibody for , 3 hours at RT followed by NorthernLightsTM 557 donkey anti-sheep lgG at RT for 1 hour. Between each step, cells were washed with PBS containing 0.1% BSA. , Flow cytometry: 50ug/ml and add 10il of the diluted solution to 1-2.5 x 10e5 cells in a total reaction volume not exceeding 200ul. The binding of unlabeled antibodies may be visualized by adding 10il of a 25ug/ml stock solution of a secondary developing reagent such as anti-sheep lgG conjugated to a fluorochrome. , Direct ELISA: 0.5-1.0ug/ml with the appropriate secondary reagents to detect human Notch-1. The detection limit is ~0.2 ng/well. , Optimal dilutions should be determined by each laboratory for each application. Storage and Stability: Lyophilized powder may be stored at -20°C. Reconstitute to nominal volume by adding sterile buffer. Aliquot and store at -20°C. Reconstituted product is stable for 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.