ADAM-9 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ADAM-9 on SDS-PAGE and why does it run higher than predicted?

The ADAM-9 (Q13443) polypeptide backbone predicts ~84 kDa from sequence alone, but this HEK293-expressed recombinant typically migrates at 90–100 kDa under reducing SDS-PAGE conditions due to N-linked glycosylation. The glycan contribution is authentic — HEK293 cells reproduce the mammalian glycosylation pattern of the native enzyme — so this apparent MW shift is expected and not indicative of aggregation or degradation. Confirm the band identity with the matched rabbit polyclonal RP-ADAM9, which is validated for Western blot against this exact recombinant.

Which isoform or processing form is this ADAM-9 recombinant — full-length, prodomain-cleaved, or catalytic domain only?

This recombinant corresponds to the mature, prodomain-cleaved ectodomain of human ADAM-9 (UniProt Q13443). The prodomain is removed during processing in HEK293 cells, yielding an active enzyme that retains the catalytic metalloprotease domain, disintegrin domain, and cysteine-rich/EGF-like regions — but not the transmembrane segment or cytoplasmic tail. This configuration is relevant for solution-phase activity assays and integrin-binding studies. Researchers requiring full-length transmembrane-anchored ADAM-9 should note this distinction when designing cell-based experiments.

What substrates does ADAM-9 cleave and which fluorogenic peptide substrate works best for activity assays?

ADAM-9 is a zinc-dependent metalloprotease (EC 3.4.24.-) with confirmed sheddase activity toward substrates including HB-EGF, Delta-1, and meltrin-β targets. For fluorogenic activity assays, the Mca-KPLGL-Dpa-AR-NH2 peptide (or equivalent FRET-quenched MMP/ADAM substrates with a P1′ leucine) is commonly employed at 10–50 µM in assay buffer. A minimum of 50–200 ng of this recombinant per reaction typically yields a measurable fluorescence signal within 30–60 minutes at 37°C. Always run a no-enzyme blank and a TAPI-0 or GM6001 inhibitor control to confirm zinc-dependent activity.

What assay buffer conditions should I use for ADAM-9 activity and IC50 experiments?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂ — is compatible with direct use in activity assays after dilution to working concentration. For IC50 determinations, dilute into assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 0.05% Brij-35 to minimize surface adsorption). Final glycerol should stay below 1% to avoid viscosity artifacts on fluorescence readings. Pre-incubate enzyme with inhibitor for 15–30 minutes at room temperature before adding substrate to allow equilibrium binding, particularly for slow-binding inhibitors.

What starting concentration of recombinant ADAM-9 is recommended for a fluorogenic protease activity assay?

A practical starting point is 100–200 ng per 100 µL reaction (roughly 1–2 nM based on ~90 kDa apparent MW), titrated against 20 µM fluorogenic substrate. At this range, initial velocity is typically linear for 30–45 minutes, allowing reliable Km and kcat determination. If signal is low, scale up to 500 ng before increasing substrate concentration, as substrate excess can introduce inner-filter effect artifacts in fluorescence-based formats. Purity is >90% by SDS-PAGE, so protein concentration measurements by A280 or BCA reliably reflect active enzyme content with minimal correction.

Can I use ADAM-9 (Recombinant) as a positive control for Western blot with the matched RP-ADAM9 antibody?

Yes — this is the primary validated use case for co-purchasing REC-ADAM9 with RP-ADAM9. Load 20–50 ng of recombinant ADAM-9 per lane under reducing conditions; expect a band at 90–100 kDa. RP-ADAM9 (rabbit polyclonal) has been validated against this exact recombinant in Western blot, giving a clean signal at 1:1,000–1:2,000 dilution with standard HRP-conjugated secondary antibodies. Because the recombinant and antibody originate from the same TPB development pipeline, this pairing eliminates the uncertainty of cross-validating reagents from different suppliers — particularly relevant for antibody validation experiments.

How much recombinant ADAM-9 should I load as a positive control alongside cell lysate samples on a Western blot?

Load 20–50 ng of REC-ADAM9 in a dedicated lane adjacent to your cell lysates. At 20 ng, RP-ADAM9 at 1:1,000 dilution produces a detectable band at 90–100 kDa without overwhelming the ECL signal relative to typical endogenous ADAM-9 expression levels in most cell lines. If your lysate lanes are from ADAM-9-overexpressing cells, match the recombinant load accordingly to keep band intensities comparable. The recombinant runs as a single clean band under reducing SDS-PAGE, making it straightforward to distinguish from higher-MW non-specific bands that occasionally appear with crude lysates.

How should I store and handle recombinant ADAM-9 to preserve activity after receiving it, and what is the shelf life?

Upon receipt, briefly centrifuge the vial and store immediately at -20°C in the supplied single-use aliquots. Avoid repeated freeze-thaw cycles — each cycle can reduce metalloprotease activity by 10–20% depending on protein concentration and glycerol buffering. The 10% glycerol and 5 mM CaCl₂ in the storage buffer are critical for maintaining zinc-site integrity; do not dialyze these components out before assays. Shelf life is 12 months from date of manufacture when stored correctly. For experiments requiring diluted working stocks, prepare fresh dilutions in assay buffer on the day of use and keep on ice.