ADAM-33 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ADAM-33 on SDS-PAGE or Western blot?

The full-length ADAM-33 protein (UniProt Q9BZ11) has a calculated MW of ~90 kDa from its 813-amino-acid sequence. However, this HEK293-expressed recombinant carries mammalian N-linked glycosylation, which typically shifts the apparent MW on SDS-PAGE to ~100–120 kDa under reducing conditions. The recombinant preparation represents the processed ectodomain after prodomain removal, so you may observe a predominant band in the 85–95 kDa range depending on the extent of glycosylation and any autocatalytic processing. Confirm identity with our matched antibody RP-ADAM33, which recognizes this processed form cleanly on Western blot.

What isoform or processing state is this recombinant ADAM-33 — full-length, prodomain-cleaved, or ectodomain?

This preparation corresponds to the processed, prodomain-cleaved ectodomain form of ADAM-33, which is the catalytically competent species. ADAMs are synthesized as inactive zymogens; prodomain removal — either autocatalytically or via furin-like proprotein convertases in the secretory pathway — is required for metalloprotease activity. Because expression is in HEK293 cells, intracellular proprotein convertase activity during trafficking generates the active form. The transmembrane and cytoplasmic regions are not present in this soluble recombinant. This is the most experimentally relevant form for biochemical activity assays and for use as an antigen loading control.

What substrates does ADAM-33 cleave, and which peptide substrate works best for an in vitro activity assay?

ADAM-33 is a zinc-dependent sheddase with validated activity toward extracellular matrix components and cell-surface proteins implicated in airway remodeling. For in vitro fluorescence-based activity assays, a generic ADAM-family FRET peptide substrate such as Mca-PLAQAV-Dpa-RSSSR-NH2 (or the equivalent Mca-KPLGL-Dpa substrate) is commonly employed at 10–20 µM final concentration. Run assays in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2 — which matches the storage buffer supplied with this lot — and include 0.01% Brij-35 to minimize enzyme adsorption to tube walls. Monitor fluorescence at Ex/Em 320/405 nm.

What starting enzyme concentration should I use for an ADAM-33 activity assay, and how do I optimize it?

A practical starting point is 50–200 nM recombinant ADAM-33 in a 50–100 µL reaction with 10–20 µM fluorogenic substrate. Titrate enzyme from 25–400 nM while keeping substrate at a fixed, sub-saturating concentration to confirm linear velocity before committing to inhibitor studies. Ensure the reaction volume includes 5 mM CaCl2 and 0.5–1 mM APMA (4-aminophenylmercuric acetate) is NOT required here, as prodomain removal is already complete. Include a no-enzyme blank and a broad-spectrum hydroxamate control (e.g., GM6001 at 10 µM) to define background and maximal inhibition bounds for any IC50 determination.

What buffer conditions are optimal for ADAM-33 recombinant metalloprotease activity, and can I add EDTA or DTT?

ADAM-33 requires free zinc at its catalytic site and calcium for structural stability. The supplied storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2) is compatible directly with activity assays after appropriate dilution. Do not add EDTA or EGTA — even 1 mM chelator will rapidly inactivate the enzyme by stripping active-site zinc. Likewise, avoid DTT or β-mercaptoethanol above 0.1 mM; disulfide bonds in the disintegrin and cysteine-rich domains are functionally important. If your downstream assay requires a reducing environment, substitute with 0.1 mM TCEP as a milder alternative and validate retention of activity.

Can I use recombinant ADAM-33 as a positive control for Western blot with the RP-ADAM33 antibody?

Yes — this is one of the primary design rationales for pairing REC-ADAM33 with RP-ADAM33. The rabbit polyclonal antibody (RP-ADAM33; /anti-adam-33-rabbit-polyclonal-antibody) was raised and validated against the same recombinant ectodomain, so specificity is confirmed rather than predicted. Load 10–50 ng of REC-ADAM33 per lane alongside your cell lysate samples; at this range, expect a clear band at ~100–120 kDa (glycosylated form) with RP-ADAM33 at 1:1,000–1:2,000 dilution. This pairing is particularly useful when running ADAM-33 Western blots on airway epithelial or smooth muscle cell lysates where endogenous expression is low and a clean positive control is essential.

How much recombinant ADAM-33 should I load as a positive control for Western blot, and what band should I expect?

Load 10–50 ng per lane for a strong, clean signal with RP-ADAM33 (rabbit polyclonal, SKU: RP-ADAM33) at a 1:1,000 antibody dilution. The primary band should appear at ~100–120 kDa, reflecting HEK293-derived glycosylation of the processed ectodomain. Purity is >95% by SDS-PAGE, so satellite bands should be minimal; any faint lower band near 85 kDa may represent a less-glycosylated species or a secondary processing product and does not indicate degradation. Run reduced, denaturing conditions (standard Laemmli with β-ME or DTT) for sharpest band resolution. Do not exceed 100 ng per lane, as overloading can cause smearing with this glycoprotein.

How should I store recombinant ADAM-33 after receipt, and what is its shelf life once thawed?

Upon receipt, spin briefly and store at -20°C in single-use aliquots — the 10% glycerol in the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2) acts as a cryoprotectant and prevents ice-crystal damage. Avoid repeated freeze-thaw cycles; each cycle can reduce metalloprotease activity by 15–30% based on internal QC comparisons. Once thawed, keep on ice and use within 4–8 hours for activity assays. For Western blot-only applications (denatured protein), a thawed aliquot can be kept at 4°C for up to 48 hours without significant band quality loss. Long-term stability at -80°C is acceptable but -20°C is standard for routine use.