ADAM-19 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ADAM-19 on SDS-PAGE or Western blot?

The full-length human ADAM-19 protein (UniProt Q9H013) has a predicted molecular weight of ~109 kDa from its 955-amino-acid sequence, but the mature processed form — lacking the signal peptide and prodomain — typically runs at approximately 85–95 kDa under reducing SDS-PAGE conditions. Glycosylation from the HEK293 expression system adds heterogeneous mass, so expect a diffuse band rather than a sharp single band. Under non-reducing conditions, disulfide-linked species may shift the apparent MW further. Confirm identity with matched antibody RP-ADAM19 (/anti-adam-19-rabbit-polyclonal-antibody).

Which processing form of ADAM-19 is this recombinant — prodomain-intact zymogen or mature active enzyme?

This recombinant is produced as an active enzyme: the prodomain has been removed, leaving the mature metalloprotease, disintegrin, cysteine-rich, and EGF-like ectodomain region. The zinc-dependent catalytic domain is therefore exposed and enzymatically competent without any additional activation step. If your assay requires a zymogen control, note that this preparation is not suitable for that purpose. The HEK293 expression system ensures proper disulfide-bond formation within the cysteine-rich domain, which is important for ectodomain shedding activity against neuregulin substrates.

What substrates does ADAM-19 cleave and what peptide substrate can I use in a fluorescent activity assay?

ADAM-19 cleaves the ectodomains of beta-type neuregulin isoforms (e.g., NRG-1β) and has documented activity against alpha-2 macroglobulin. For a fluorescent activity assay, the generic ADAM-family quenched fluorescent substrate Mca-KPLGL-Dpa-AR-NH2 (or the closely related Mca-PLAQAV-Dpa-RSSSR-NH2) is commonly used and provides measurable signal with ADAM family metalloproteases. Run the reaction in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2 at 37°C. Validate substrate cleavage by running a no-enzyme control and a EDTA-inhibited control (10 mM EDTA) in parallel to confirm zinc-dependence.

What buffer conditions and starting enzyme concentration should I use for an ADAM-19 activity assay?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol — is compatible with direct use in activity assays; dilute into the same buffer (minus glycerol if signal interference is a concern above 0.5%). For initial titration, start at 50–200 nM enzyme with 10–50 µM fluorescent substrate. Include 0.05% Brij-35 or 0.1% BSA to minimize non-specific surface adsorption at these concentrations. Glycerol carry-over above 1% final concentration can modestly reduce kcat; keep dilutions ≥1:10 from stock to avoid this effect.

How do I set up an IC50 inhibition assay with recombinant ADAM-19 and a test compound?

Use 50–100 nM ADAM-19 with substrate at or near Km (empirically determined per fluorescent substrate used, typically 10–30 µM for Mca-peptides). Pre-incubate enzyme with inhibitor for 30 minutes at 37°C before adding substrate — ADAM metalloprotease active sites can be slow-binding targets. EDTA (10 mM) and marimastat (broad-spectrum hydroxamate, 1 µM) serve as positive inhibition controls. Run each inhibitor concentration in triplicate and fit to a four-parameter logistic curve. Purity >90% by SDS-PAGE ensures that IC50 values are not artifactually elevated by inactive protein.

Can I use REC-ADAM19 as a positive control for Western blot with the matched antibody RP-ADAM19?

Yes — this is one of the primary use cases for pairing REC-ADAM19 with RP-ADAM19 (/anti-adam-19-rabbit-polyclonal-antibody). Load 25–100 ng of recombinant protein per lane alongside your cell lysate samples. At 25 ng you should see a clear band at ~85–95 kDa under reducing conditions that comigrates with endogenous ADAM-19 in positive-control cell lines. RP-ADAM19 is validated for Western blot with this recombinant in-house, so it provides a definitive size and signal reference for antibody titration experiments and lot-to-lot consistency checks.

How much recombinant ADAM-19 should I load per lane to validate antibody RP-ADAM19 on Western blot?

For a standard antibody validation Western blot, a two-fold dilution series of 10 ng, 25 ng, 50 ng, and 100 ng per lane works well. This range spans the linear detection window for most HRP-conjugated secondaries and allows you to confirm both sensitivity and specificity of RP-ADAM19. Use reducing sample buffer; the disulfide-linked ectodomain will partially shift under non-reducing conditions, which can complicate band interpretation during validation. Run alongside a lysate lane so you can confirm matched migration of the endogenous and recombinant bands.

How should I store and handle recombinant ADAM-19 to maintain activity after receipt?

Upon receipt, REC-ADAM19 is shipped on dry ice in single-use aliquots in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol. Store at -20°C immediately; avoid repeated freeze-thaw cycles, which denature the zinc-binding active site and degrade disulfide-integrity in the cysteine-rich domain. For working dilutions, prepare in the same buffer supplemented with 0.1% BSA to stabilize at low concentrations (below ~10 nM). Diluted working stocks are stable for up to 24 hours at 4°C. Do not store diluted enzyme at -20°C — the reduced glycerol concentration provides insufficient cryoprotection.