ADAMTS-16 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ADAMTS-16 on SDS-PAGE or Western blot?

The full-length human ADAMTS-16 protein (UniProt Q8TE57) encodes 1,224 amino acids, giving a predicted unmodified MW of ~137 kDa. However, because this recombinant is produced in HEK293 cells, N-linked glycosylation adds significant mass — expect a diffuse or slightly smeared band running between ~150–175 kDa under reducing SDS-PAGE conditions. The pro-domain may or may not be cleaved depending on the specific construct; confirm the exact processed form in the certificate of analysis. When using our matched antibody RP-ADAMTS16 for Western blot, load 20–50 ng of this recombinant as a positive control lane.

Is recombinant ADAMTS-16 the pro-form or the mature processed enzyme?

This preparation is produced as the mature, pro-domain-processed form. HEK293 cells support furin-like proprotein convertase activity in the secretory pathway, which cleaves the ADAMTS-16 pro-domain at the canonical RXXR motif prior to secretion. The secreted, processed enzyme is what is collected and supplied. Lot-specific processing efficiency is confirmed by SDS-PAGE and reported on the certificate of analysis. Researchers studying pro-domain inhibition or zymogen activation kinetics should note this distinction and consider whether a pro-form construct is more appropriate for their experimental design.

What substrates does ADAMTS-16 cleave and which is best for an in vitro activity assay?

ADAMTS-16 is classified as EC 3.4.24.- and has documented aggrecanase activity, cleaving the Glu373–Ala374 bond in the interglobular domain of aggrecan — the canonical ADAMTS substrate. For a fluorescent in vitro activity assay, the FRETS-aggrecan substrate (Mca-TEGE↓ARGS-Dpa) is the most widely used option and is compatible with this enzyme. Alternatively, the generic metalloprotease substrate Mca-KPLGL-Dpa-AR-NH2 can provide baseline activity readout. Run reactions in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35 at 37°C; the supplied storage buffer is directly compatible.

What starting concentration of recombinant ADAMTS-16 should I use in a protease activity assay?

For fluorescence-based substrate cleavage assays, a starting concentration of 1–10 nM recombinant ADAMTS-16 is a practical range to bracket initial velocity conditions. With the Mca-TEGE↓ARGS-Dpa substrate, use 5–25 µM substrate and 2–5 nM enzyme for Km determination runs. Because ADAMTS family members are calcium-dependent, confirm that your assay buffer contains at least 5 mM CaCl2 — the supplied storage buffer already includes this. Titrate enzyme concentration empirically if switching to a non-canonical substrate or if working with tissue-derived inhibitor-containing lysates where endogenous TIMP levels may suppress apparent activity.

How does buffer composition affect ADAMTS-16 activity and can I dilute it into my own assay buffer?

ADAMTS-16 is a zinc metalloprotease with strict calcium dependence; activity drops sharply if free Ca²⁺ falls below ~1 mM. The supplied storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol) is designed to preserve both stability and cofactor coordination. When diluting into your assay buffer, ensure it contains at least 2–5 mM CaCl2 and is free of EDTA or EGTA, which will chelate zinc and calcium and abolish activity. A low concentration of non-ionic detergent (0.01–0.05% Brij-35 or Tween-20) helps prevent surface adsorption at sub-nanomolar working concentrations.

Can I use recombinant ADAMTS-16 as a positive control for Western blot with the RP-ADAMTS16 antibody?

Yes — this is the primary validated use case for pairing REC-ADAMTS16 with RP-ADAMTS16. Load 20–50 ng of recombinant ADAMTS-16 per lane alongside your cell lysate or conditioned media samples. The rabbit polyclonal RP-ADAMTS16 is validated for Western blot and will produce a clear band at ~150–175 kDa (glycosylated form) that serves as an unambiguous size and identity reference. This is especially useful when working with samples where endogenous ADAMTS-16 expression is low or variable. See the RP-ADAMTS16 product page (/anti-adamts-16-rabbit-polyclonal-antibody) for recommended antibody dilutions and blocking conditions.

How much recombinant ADAMTS-16 should I load to validate the RP-ADAMTS16 antibody specificity in IHC optimization?

For antibody validation purposes — particularly dot-blot or slot-blot controls run alongside IHC — 5–20 ng of REC-ADAMTS16 per spot is sufficient to confirm RP-ADAMTS16 binding specificity before committing to tissue sections. For Western blot specificity controls, 20–50 ng per lane under reducing conditions works reliably. RP-ADAMTS16 is validated for Western blot per TPB (immunohistochemistry (IHC) and immunofluorescence (IF) validation in progress)'s standard validation panel. Loading a known quantity of REC-ADAMTS16 as a parallel positive control is the most direct way to confirm antibody lot-to-lot consistency, particularly for IHC protocols where antigen retrieval conditions can influence apparent signal intensity.

What are the storage and handling requirements for recombinant ADAMTS-16 to maintain activity after receipt?

Upon receipt, spin briefly to collect the solution, then aliquot into single-use volumes at your intended working concentration before freezing at -20°C. The supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol) provides cryo-protection; do not remove glycerol prior to long-term storage. Avoid repeated freeze-thaw cycles — each cycle can reduce recoverable metalloprotease activity by 10–20% and promote aggregation. For experiments running across multiple days, keep thawed aliquots on ice and use within 24–48 hours. Do not dilute to below ~0.1 µg/mL in the absence of a carrier protein, as adsorptive losses become significant at that concentration.