Matriptase-1 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant Matriptase-1 on SDS-PAGE or Western blot?

The full-length human Matriptase-1 (ST14, UniProt Q9Y5Y6) has a predicted molecular weight of ~95 kDa from its 855-amino-acid sequence. Because this recombinant is produced in HEK293 cells, N-linked glycosylation adds appreciable mass; the apparent MW on reducing SDS-PAGE typically runs at 110–125 kDa. Under non-reducing conditions, disulfide-linked forms may shift further. If your blot shows a band in the 110–125 kDa range with diffuse trailing — consistent with glycosylation heterogeneity — that is the expected result, not a degradation artifact.

Which isoform or processing form of Matriptase-1 is present in this recombinant preparation?

This preparation represents the extracellular region of human Matriptase-1, expressed as a soluble recombinant fragment encompassing the SEA domain, two CUB domains, four LDLRA repeats, and the C-terminal serine protease domain. The transmembrane anchor and cytoplasmic tail are excluded. The protease domain is supplied in its active, zymogen-processed form — consistent with the autocatalytic activation that occurs physiologically. Single-chain and two-chain (activated) species may both be present; the preparation is validated for enzymatic activity against synthetic fluorogenic substrates.

What substrates does recombinant Matriptase-1 cleave, and how do I set up a fluorogenic activity assay?

Matriptase-1 exhibits trypsin-like activity, preferentially cleaving after Arg or Lys at the P1 position. For fluorogenic assays, Boc-Gln-Ala-Arg-AMC and Tosyl-Gly-Pro-Arg-AMC (Tos-GPR-AMC) are well-validated substrates. A standard starting point: 50–100 nM recombinant Matriptase-1 in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) with 100–200 µM fluorogenic substrate at 37°C; read at ex/em 380/460 nm. Confirm linearity over 30–60 minutes before adding inhibitors. Avoid including reducing agents (e.g., DTT >1 mM), which can disrupt disulfide-dependent domain integrity.

What buffer conditions are optimal for Matriptase-1 activity assays, and does glycerol in the storage buffer interfere?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with direct use in activity assays when the enzyme is diluted at least 1:10 into assay buffer, bringing glycerol to ≤1%, which does not meaningfully affect trypsin-like serine protease activity. Matriptase-1 is active across pH 6.5–8.5, with an optimum near pH 7.5. Avoid EDTA (it can chelate structurally relevant metal ions in CUB domains) and serine protease inhibitors such as PMSF or AEBSF in any pre-incubation steps. Calcium (1–2 mM) is not required but is tolerated.

What starting enzyme concentration should I use for Matriptase-1 inhibitor IC50 assays?

For inhibitor titration experiments, 10–50 nM recombinant Matriptase-1 is a practical starting range that gives robust signal with 100–200 µM Tos-GPR-AMC substrate while remaining below typical Ki values for well-characterized inhibitors like HAI-1/Kunitz domain peptides (Ki ~0.5–5 nM) or small-molecule benzamidine derivatives. At these concentrations, tight-binding inhibitor corrections (Morrison equation) may be required if Ki approaches enzyme concentration. Always pre-incubate enzyme with inhibitor for 15–30 minutes at room temperature before adding substrate to allow equilibrium binding.

Can I use recombinant Matriptase-1 as a positive control for Western blot with the RP-Matriptase1 rabbit polyclonal antibody?

Yes — this is a primary validated use case. The matched antibody RP-Matriptase1 (/anti-matriptase-1-rabbit-polyclonal-antibody) was raised against the same recombinant antigen and is guaranteed compatible for Western blot positive control use. Load 50–100 ng of recombinant Matriptase-1 per lane on a 10% SDS-PAGE gel; expect a band at 110–125 kDa under reducing conditions. Use RP-Matriptase1 at 1:500–1:2,000 dilution as a starting point. This pairing is also well-suited for antibody lot-to-lot validation and as a quantitation reference when building a standard curve.

How much recombinant Matriptase-1 should I load for a Western blot positive control lane?

50–100 ng per lane is sufficient for a clear, clean band with the matched RP-Matriptase1 antibody on standard PVDF or nitrocellulose membranes. At 50 ng, signal is detectable without overwhelming nearby lanes; 100 ng is more appropriate when the antibody is used at higher dilution (1:2,000) or when the blot is stripped and re-probed. Dilute the recombinant directly into Laemmli sample buffer, boil for 5 minutes, and load immediately. Because the preparation is >90% pure by SDS-PAGE, a single dominant band is expected with minimal background.

How should I store and handle recombinant Matriptase-1 to preserve enzymatic activity over time?

Store at -20°C in the single-use aliquots as supplied. Repeated freeze-thaw cycles progressively reduce specific activity — even two additional cycles can cause measurable loss in Tos-GPR-AMC cleavage rate. For experiments spanning multiple days, thaw one aliquot, keep on ice, and use within 24 hours. Do not dilute working stocks in plain water; use 50 mM Tris-HCl pH 7.5, 150 mM NaCl with 0.1% BSA as a carrier to prevent surface adsorption at low concentrations (<10 nM). Under proper conditions, activity is stable for at least 12 months from the date of manufacture.