ADAMTS-19 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ADAMTS-19 on SDS-PAGE and Western blot?

ADAMTS-19 (UniProt Q8TE59) has a predicted unprocessed molecular weight of ~134 kDa based on its 1,213-amino acid sequence. The HEK293-expressed recombinant carries mammalian N- and O-linked glycosylation, so the apparent band on reducing SDS-PAGE typically migrates at 150–165 kDa — meaningfully higher than the sequence-predicted mass. If you are using RP-ADAMTS19 for Western blot, load this recombinant as your positive control and expect the band in that 150–165 kDa window, not at the theoretical unglycosylated weight.

What processing form is the recombinant ADAMTS-19 — full-length or mature active form, and is the prodomain removed?

The recombinant is produced in HEK293 cells and secreted into conditioned medium, which allows endogenous furin-like proprotein convertases in the secretory pathway to process the prodomain cotranslationally, mirroring physiological maturation. The product therefore represents the mature, prodomain-processed form retaining the catalytic metalloprotease domain, disintegrin-like domain, cysteine-rich domain, spacer domain, and ancillary thrombospondin type-1 repeats. Purity is >90% by SDS-PAGE. If your experiment requires the zymogen form, this preparation is not suitable.

What substrates can I use to measure recombinant ADAMTS-19 proteolytic activity in a fluorescence assay?

ADAMTS-19 is a zinc metalloprotease (EC 3.4.24.-) with broad ECM substrate relevance. For fluorometric activity assays, the generic metalloprotease FRET substrate Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (commonly used for ADAMTS family members) provides a practical starting point; monitor cleavage at Ex/Em 320/405 nm. Proteoglycan-derived peptides such as aggrecan IGD-region sequences are more physiologically relevant if substrate selectivity matters for your model. Include 5 mM CaCl2 in the reaction buffer — already present in the supplied storage buffer — as calcium is required for catalytic activity.

What is the recommended activity assay buffer for ADAMTS-19 recombinant protein and do I need to add zinc or calcium?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2. For fluorometric or cleavage assays, dilute directly into a reaction buffer of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35. Zinc is structurally incorporated into the metalloprotease active site and does not need to be supplemented; adding exogenous ZnCl2 above low micromolar concentrations can paradoxically inhibit activity. Keep reaction pH between 7.0–7.8 — activity drops sharply outside this range for ADAMTS family metalloproteases.

What starting concentration and dilution range should I use for recombinant ADAMTS-19 in enzyme activity dose-response experiments?

For initial activity titrations, a working range of 0.5–10 nM (roughly 0.07–1.4 µg/mL based on ~140 kDa apparent MW) covers the linear response window for most FRET-substrate assays. Start at 2 nM as a single-point screen; if signal is saturated, dilute down in the assay buffer rather than in the storage glycerol-containing buffer to avoid substrate interference. For inhibitor IC50 determinations, fix enzyme at 1–2 nM (near the Km-relevant concentration) and titrate inhibitor across 8–10 points. Always pre-incubate enzyme at 37°C for 10 min before adding substrate.

Can I use recombinant ADAMTS-19 as a positive control for Western blot with the RP-ADAMTS19 rabbit polyclonal antibody?

Yes — this is the primary validated use case for pairing REC-ADAMTS19 with RP-ADAMTS19. The matched rabbit polyclonal antibody (RP-ADAMTS19) was raised and validated in the same laboratory, and compatibility between antigen and antibody is confirmed. Load 50–100 ng of recombinant per lane on a 4–12% Bis-Tris gel under reducing conditions; expect a band at 150–165 kDa. This pairing is particularly useful when setting up ADAMTS-19 detection in tissue or cell lysates for the first time, giving you an unambiguous size reference and confirming antibody performance on your gel system.

How much recombinant ADAMTS-19 should I load as a positive control, and what antibody dilution works with RP-ADAMTS19?

Load 50–100 ng per lane for a clean positive control band with RP-ADAMTS19; going above 200 ng risks smearing given the glycoprotein heterogeneity. For the antibody, a starting dilution of 1:1,000–1:2,000 in 5% non-fat milk/TBST works for most standard ECL detection systems. If background is high, switch the blocking buffer to 5% BSA/TBST, which is generally preferable for phospho-independent targets including metalloproteases. RP-ADAMTS19 is validated for Western blot; cross-reactivity with other ADAMTS family members has not been validated, so it should not be assumed.

How should I store recombinant ADAMTS-19 and what is the shelf life after thawing?

Store at -20°C in the supplied single-use aliquots; do not refreeze after thawing. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2) is formulated for long-term stability, and glycerol prevents freeze-induced aggregation. Once thawed, keep on ice and use within 4–8 hours; metalloprotease activity declines with extended time at room temperature due to autoproteolysis. For experiments requiring dilution below ~0.1 µg/mL, add carrier protein (0.1% BSA) to the dilution buffer to prevent adsorptive losses to tube walls.