ADAMTS-8 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ADAMTS-8 on SDS-PAGE or Western blot?

The full-length human ADAMTS-8 polypeptide (889 aa, UniProt Q9UP79) has a predicted unglycosylated MW of ~99 kDa. Because this recombinant is expressed in HEK293 cells and carries native N-linked glycosylation, the apparent MW on reducing SDS-PAGE typically runs at 110–130 kDa. Autocatalytic or furin-mediated propeptide removal during secretion may also generate a processed ~95–105 kDa catalytic form in the preparation. Running a reducing gel is recommended; non-reducing conditions can produce higher-MW disulfide-linked species that shift migration further.

What processed or mature form does recombinant ADAMTS-8 correspond to, and is the prodomain removed?

HEK293-expressed ADAMTS-8 undergoes prodomain processing consistent with furin-type proprotein convertase cleavage at the conserved RXXR motif upstream of the catalytic domain — the same processing that occurs in vivo. The commercial preparation therefore contains predominantly the processed, active form retaining the metalloproteinase, disintegrin-like, thrombospondin type-1 repeat, cysteine-rich, spacer, and C-terminal thrombospondin repeat domains. A minor band corresponding to the unprocessed zymogen may be visible at >130 kDa under reducing SDS-PAGE. Purity is >90% by SDS-PAGE across both species.

What substrates does ADAMTS-8 cleave and how do I set up a fluorescent activity assay?

ADAMTS-8 is an aggrecanase with documented activity against the aggrecan interglobular domain (IGD), cleaving at the Glu373–Ala374 bond. For fluorescent activity assays, the quenched fluorescent peptide substrate Mca-TESE↓ESRGAI-Dpa (aggrecanase FRET substrate, available from multiple suppliers) is a practical starting point. Use a final substrate concentration of 10–20 µM in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 0.05% Brij-35) at 37°C. Monitor fluorescence at Ex/Em 320/405 nm. Pilot enzyme concentrations of 0.5–5 nM to establish linearity before running inhibitor panels.

What is the recommended starting concentration of recombinant ADAMTS-8 for an aggrecanase activity assay?

A starting range of 1–10 nM recombinant ADAMTS-8 is appropriate for Mca-based FRET assays in standard 100 µL reaction volumes. Begin at 5 nM and confirm that initial velocity is linear over at least 30 minutes and that substrate turnover remains below 10% at the assay endpoint to satisfy steady-state kinetic assumptions. If you are screening inhibitors, a final enzyme concentration at or just below the apparent Km of the substrate helps maximize assay sensitivity. Excess zinc or EDTA in sample diluents will inhibit activity — verify that your inhibitor vehicle does not chelate Ca²⁺.

What buffer conditions are optimal for recombinant ADAMTS-8 enzyme activity, and can I dilute into PBS?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 10% glycerol — is also a suitable working buffer. If your assay format requires dilution into PBS, supplement with at least 5 mM CaCl₂ and 0.05% BSA or 0.01% Tween-20 to prevent adsorption to low-binding tubes at sub-nanomolar concentrations. Avoid phosphate buffers containing high EDTA or EGTA; even low micromolar concentrations of metal chelators abolish zinc-metalloproteinase activity. Bring aliquots to room temperature just before use and do not re-freeze after thawing.

Can I use recombinant ADAMTS-8 as a positive control for Western blot with the matched RP-ADAMTS8 antibody?

Yes — this is a validated use case. The matched rabbit polyclonal antibody RP-ADAMTS8 (/anti-adamts-8-rabbit-polyclonal-antibody) was raised and validated in the same program as REC-ADAMTS8, making the recombinant the natural positive control. Load 20–50 ng of recombinant ADAMTS-8 per lane under reducing conditions; the antibody should detect the processed ~110–130 kDa glycoprotein band without cross-reactive background. If you are confirming antibody specificity in cell lysates alongside the recombinant lane, this pairing also satisfies the "matched positive control" requirement common in antibody validation frameworks.

How much recombinant ADAMTS-8 should I load for a Western blot positive control lane?

Load 20–50 ng per lane as a starting point when using RP-ADAMTS8 at a 1:500–1:2000 dilution in 5% non-fat milk/TBST. At 50 ng you should see a strong band in the 110–130 kDa region after standard ECL detection; 20 ng is typically sufficient with high-sensitivity chemiluminescence substrates. Include a negative control lane (buffer only) alongside the recombinant lane to confirm that any HEK293 host-cell protein carry-through does not produce confounding bands in your region of interest. Running a titration (10, 25, 50 ng) on your first use establishes the dynamic range for your specific detection system.

How should I store and handle recombinant ADAMTS-8 to maintain enzyme activity over time?

Store at -20°C in single-use aliquots immediately upon receipt. The 10% glycerol in the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂) provides cryoprotection and supports stability for at least 12 months at -20°C when freeze-thaw is avoided. Aliquot to volumes matching your typical single-experiment usage — repeated freeze-thaw cycles measurably reduce metalloproteinase specific activity. On the day of use, thaw on ice, spin briefly at 10,000 × g to collect any condensate, and keep on ice during setup. Do not store working dilutions; discard unused thawed material.