| The amyloid b-peptide (Ab) of 39 to 43 amino acids is constitutively produced in brain upon proteolysis of the b-amyloid precursor protein (APP) and exists as fragments of 40, 42 and 43 amino acids (Ab40, Ab42, Ab43). In the young and healthy humans, the secreted Ab is rapidly catabolized before it can be deposited in the brain. However, upon aging or the onset of familial Alzheimers disease, alterations in either synthesis or degradation/clearance of Ab may contribute to amyloid depositions in the brain. Ab carries cleavage sites for a number of in vivo and in vitro proteases like cathepsin D and M-13 metalloproteases. The M-13 family comprises several zinc-dependent metalloproteases like DINE, PHEX, KELL, ECE, XCE, neprilysin (NEP) and neprilysin-like proteases (NEPL-a, NEPL-b, NEPL-g). NEP [variously termed as neutralendopeptidase-24.11 (NEP), neprilysin, enkephalinase, EC3.4.24.11, common acute lymphoblastic leukemia antigen (CALLA), CD10] is the key in vivo enzyme degrading biopeptides Ab, substance-P and enkephalin in brain, atrial natriuretic peptide, bradykinin and endothelin-1 in kidney. It is a 97kD ectoenzyme (mouse, rat, human 750aa) with a large extracellular domain containing its catalytic site, which can degrade Ab on cell surface. NEP is ubiquitous but its high expression in brain is restricted to striatum, olfactory tubercle, substantia nigra, choroids plexes, endopeduncular nucleus, pontine nucleus, and cerebellum and in many peripheral tissues, particularly in brush-border membranes. NEP is also expressed in soluble form in human plasma and cerebrospinal fluid. NEP can degrade both synthetic and cell secreted Ab40 and Ab42 and may be a good therapeutic target for the treatment of Alzheimers disease. Source: Mouse NEP protein (Tyr45-Trp743, total of 699 residues) was expressed in baculovirus cell line Sf21. The purified protein migrates as ~90kD in SDS-PAGE due to glycosylation. Applications: Suitable for use in Western Blot as a positive control for N2030-21 & N2030-22. This preparation is not biologically inactive. It is not suitable for ELISA or other applications where native protein is required. Other applications not tested. Recommended Dilution: Western Blot: Load 10ul/lane, If the product has been stored for several weeks, then it may be preferable to add 5ul of fresh 2X sample buffer per 10ul of solution prior to heating and loading on gels. Optimal dilutions to be determined by the researcher. Storage and Stability: Lyophilized powder may be stored at -20C. Stable for 12 months at -20C. Reconstitute with sterile buffer or ddH2O. Aliquot to avoid repeated freezing and thawing. Store at -20C. Reconstituted product is stable for 6 months at -20C. 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. |
