Anti-Insulin Antibody [N42]

Insulin is a 51-amino acid polypeptide assembled from A and B chains connected by disulfide bonds, and is first translated as the larger proinsulin precursor before proteolytic processing within pancreatic beta-cell secretory granules. Because the N42 epitope sits within the A/B-chain region of the hormone, the antibody also recognizes intact proinsulin, which migrates near 12 kDa under reducing SDS-PAGE conditions and appears as the dimer/misfolded band sometimes seen just above the mature monomer on western blots. Practical starting conditions used in published workflows include coating ELISA plates at 1 µg/mL in carbonate buffer pH 9.6 with 100 µL of standard or sample per well, loading 10–20 µg of total cell or tissue lysate per lane for western blot, transferring to PVDF or nitrocellulose, and probing overnight at 4 °C at dilutions between 1:1000 and 1:2000 after blocking in 5% non-fat milk in TBST. The Antibodies.com liquid formulation ships in a single vial and is ready for direct dilution into standard assay buffers. The reagent has been used to profile insulin content in beta-cell lines such as MIN6 and INS-1, in isolated human islet preparations, and to monitor beta-cell mass in streptozotocin-treated diabetic rodent models; when pairing with HRP-conjugated secondaries, omit sodium azide from working buffers to preserve peroxidase activity. The 100 µg vial provides enough material for dozens of immunoblots or multiple microplate immunoassays, depending on the chosen working dilution.