ADAM-10 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ADAM-10 on SDS-PAGE or Western blot?

The full-length human ADAM-10 polypeptide has a calculated MW of ~84 kDa, but the HEK293-expressed recombinant runs at approximately 95–105 kDa under reducing SDS-PAGE conditions due to N-linked glycosylation. The prodomain (~8 kDa) is removed during autocatalytic activation, so the mature active form typically resolves as a diffuse band in the 85–100 kDa range. If you observe a doublet, this reflects heterogeneous glycosylation — both bands are catalytically active. PNGase F treatment collapses the band to ~82 kDa, confirming glycan contribution.

Which processing form of ADAM-10 is this recombinant — zymogen or mature active enzyme?

This product is the mature, active form of human ADAM-10. During HEK293 expression and secretion, the prodomain is removed by furin-family proprotein convertases in the trans-Golgi network, yielding the catalytically competent metalloprotease. The recombinant encompasses the metalloprotease, disintegrin, cysteine-rich, and EGF-like domains. It is not a zymogen and does not require activation steps prior to use. The HEXXH catalytic motif is intact and zinc-coordinated, making this suitable for direct use in cleavage assays without any pre-activation protocol.

What substrates does recombinant ADAM-10 cleave and what fluorogenic peptide should I use for activity assays?

ADAM-10 cleaves multiple physiological substrates including APP (α-secretase site), Notch receptors (S2 cleavage), E-cadherin, and EphB2. For in vitro fluorogenic activity assays, the synthetic peptide Mca-KPLGL-Dpa-AR-NH2 (a generic MMP/ADAM substrate) is compatible, but the more ADAM-10-selective substrate Mca-PLAQAV-Dpa-RSSSR-NH2 is preferred. Excitation/emission at 320/405 nm. Run assays in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35. Starting enzyme concentration of 0.5–5 nM with 10–50 µM substrate gives reliable linear signal.

What buffer conditions are optimal for recombinant ADAM-10 activity assays and does glycerol inhibit the enzyme?

ADAM-10 is active in 25 mM Tris-HCl pH 7.5–8.0, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2) is fine for long-term stability, but dilute at least 1:5–1:10 into assay buffer before use to reduce glycerol to ≤2%, as higher concentrations modestly suppress catalytic rate. Avoid EDTA, EGTA, or 1,10-phenanthroline — these chelate the catalytic zinc and will abolish activity. DTT above 1 mM is also not recommended, as it can disrupt the disulfide architecture in the cysteine-rich domain.

What starting concentration of recombinant ADAM-10 should I use to measure inhibitor IC50 in a fluorogenic assay?

For IC50 determinations, use enzyme at a concentration well below the Km of your substrate — typically 0.5–2 nM recombinant ADAM-10 with 10 µM fluorogenic substrate. Running the enzyme at sub-Km substrate conditions satisfies the Cheng-Prusoff correction and ensures your measured IC50 approximates Ki. Include a no-enzyme blank and a DMSO vehicle control (≤0.5% DMSO final). GI254023X and INCB7839 serve as well-characterized reference inhibitors with reported ADAM-10 IC50 values of ~1–10 nM in comparable assay formats — useful for inter-run normalization.

Can I use recombinant ADAM-10 (REC-ADAM10) as a positive control for Western blot with the matched RP-ADAM10 antibody?

Yes — REC-ADAM10 and RP-ADAM10 are developed and validated together in the same lab specifically for this use case. Load 50–100 ng of recombinant ADAM-10 per lane alongside your cell lysate lanes. The RP-ADAM10 rabbit polyclonal (see /anti-adam-10-rabbit-polyclonal-antibody) detects a band at 95–105 kDa under standard reducing conditions. Use RP-ADAM10 at 1:1,000–1:2,000 dilution in 5% non-fat milk/TBST, with anti-rabbit HRP secondary at 1:5,000–1:10,000. The recombinant lane provides a clean, defined signal to confirm antibody specificity and calibrate exposure time relative to endogenous protein in your samples.

How much recombinant ADAM-10 should I load per lane as a Western blot positive control, and will it look the same as endogenous ADAM-10?

Load 50–100 ng per lane for a strong, unambiguous positive control band with RP-ADAM10. At 50 ng, signal is detectable without overloading. The recombinant runs at 95–105 kDa — the same range as endogenous ADAM-10 in HEK293, HeLa, or primary neuron lysates — so band alignment is direct and interpretable. Note that endogenous ADAM-10 may appear as a broader or slightly lower band in some cell types reflecting differences in glycan processing at individual N-glycosylation sites. Both represent correctly folded, processed protein.

How should I handle, dilute, and store recombinant ADAM-10 to maintain activity over time?

REC-ADAM10 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2. Store at -20°C in the single-use aliquots provided. Avoid repeated freeze-thaw cycles — each cycle can reduce specific activity by 10–20%. For dilution series, use assay buffer supplemented with 0.1% BSA as a carrier protein to prevent adsorption losses at low concentrations (sub-1 nM). Pre-chill tubes on ice before aliquoting. Once thawed, use within 24 hours and keep on ice. Purity is >90–95% by SDS-PAGE; endotoxin is <0.1 EU/µg by LAL, suitable for cell-based assays.