ADAM-13 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ADAM-13 on SDS-PAGE or Western blot?

The full-length ADAM-13 polypeptide (UniProt O12960) is 914 amino acids with a predicted unmodified MW of ~103 kDa. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation adds mass, and the mature secreted/shed ectodomain typically migrates at approximately 90–110 kDa under reducing conditions on SDS-PAGE — slightly diffuse due to heterogeneous glycosylation. Under non-reducing conditions the band may shift. If you see a doublet, this reflects differential glycoform populations, not contamination. Purity is confirmed >90% by silver-stained SDS-PAGE before release.

Which processing form of ADAM-13 does this recombinant represent — zymogen or active enzyme?

This product is supplied as the active, prodomain-cleaved form. During HEK293 expression, furin-like proprotein convertases cleave the prodomain autoinhibitory cap, generating the mature metalloprotease. You will not observe a higher-MW zymogen band as the predominant species. The prodomain removal follows the canonical ADAM activation mechanism documented for Xenopus ADAM-13 (adam13.L gene product). If your experiment requires the latent zymogen form as a control, that construct is not currently part of this SKU.

What substrates does ADAM-13 cleave and which is best for a fluorescent activity assay?

ADAM-13 is a zinc-dependent sheddase best characterised for cleavage of fibronectin and cadherin-11 (CDH11) ectodomains during Xenopus cranial neural crest cell migration. For quantitative in vitro activity assays, the generic ADAM/MMP fluorogenic substrate Mca-KPLGL-Dpa-AR-NH2 (or the broader ADAM substrate Mca-RPKPVE-Nval-WRK(Dnp)-NH2) provides a convenient readout at Ex/Em 320/405 nm. Confirm activity in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.01% Brij-35. A cadherin-11 ectodomain cleavage ELISA can confirm physiological substrate specificity in parallel.

What buffer conditions and starting enzyme concentration should I use for an ADAM-13 activity assay?

Use the storage buffer conditions as your assay baseline: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, with 0.01% Brij-35 added to prevent surface adsorption. A starting enzyme concentration of 50–200 nM is reasonable for fluorogenic substrate assays; titrate down if your substrate is high-affinity. Avoid EDTA or EGTA — they chelate the catalytic zinc and will abolish activity. DTT and β-mercaptoethanol should also be excluded, as free thiols can disrupt the cysteine-rich domain. Include a 1,10-phenanthroline inhibitor control (1–10 mM) to confirm metalloprotease-dependent signal.

How should I handle and dilute ADAM-13 (Recombinant) to preserve activity across multiple experiments?

The protein is shipped in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 and stored at -20°C in single-use aliquots. Do not refreeze after thawing — repeated freeze-thaw cycles progressively denature the cysteine-rich domain and reduce specific activity. For dilutions, use the same Tris-NaCl-CaCl2 base buffer supplemented with 0.1% BSA as a carrier to prevent adsorptive losses at low concentrations (<50 nM). Endotoxin is certified <0.1 EU/µg by LAL, so the preparation is compatible with cell-based assays without additional depyrogenation.

Can I use ADAM-13 (Recombinant) as a positive control for Western blot with the matched RP-ADAM13 antibody?

Yes — this is the primary validated use case for pairing REC-ADAM13 with RP-ADAM13. Load 20–50 ng of recombinant per lane; the anti-ADAM-13 rabbit polyclonal (RP-ADAM13) will detect the mature ~90–110 kDa band under standard reducing SDS-PAGE/Western conditions. Because both the recombinant and the antibody originate from the same Triple Point Biologics pipeline, antigen–antibody compatibility is guaranteed. This makes REC-ADAM13 particularly useful when validating RP-ADAM13 against endogenous ADAM-13 in Xenopus or mammalian lysates — the recombinant band serves as an unambiguous size marker and signal reference.

How much recombinant ADAM-13 should I load as a positive control to avoid saturating the RP-ADAM13 Western blot signal?

Start with a two-point titration: 20 ng and 50 ng per lane. At 20 ng, RP-ADAM13 used at a 1:1000–1:2000 dilution typically yields a clean single band at ~90–110 kDa without saturation on standard ECL film or digital imaging. Loading >100 ng per lane risks signal overflow that can mask nearby bands in multiplex blots. If you are running recombinant alongside crude Xenopus embryo lysate (where endogenous ADAM-13 is low), 50 ng provides a well-resolved reference band without overwhelming the lane. See the RP-ADAM13 product page (/anti-adam-13-rabbit-polyclonal-antibody) for full dilution and incubation conditions.

What species and isoform does this ADAM-13 recombinant correspond to, and is it suitable for mammalian cell studies?

REC-ADAM13 is derived from the Xenopus laevis adam13.L gene product (UniProt O12960, 914 aa), making it the most functionally characterised ADAM-13 species in the literature — particularly for cranial neural crest cell migration and cadherin shedding studies. Human ADAM13 is not a recognised paralogue; the closest mammalian functional orthologue is ADAM19/Meltrin-β. If your goal is studying the Xenopus developmental biology context or benchmarking substrate cleavage specificity, this recombinant is appropriate. For mammalian ADAM19-related biology, confirm cross-reactivity requirements before proceeding, as sequence identity between Xenopus ADAM-13 and human ADAM19 is moderate (~50–55% in the metalloprotease domain).