Matriptase-2 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Matriptase-2 on SDS-PAGE or Western blot?

The full-length Matriptase-2 protein (UniProt Q8IU80) has a predicted molecular weight of ~90 kDa from its 811-amino-acid sequence. However, due to N-linked glycosylation added during HEK293 expression, the recombinant protein typically migrates at approximately 95–110 kDa under reducing SDS-PAGE conditions. This glycosylation-induced shift is expected and reflects mammalian post-translational processing. If you are running a non-reducing gel, expect a slightly different migration pattern. Our >90% purity guarantee means the dominant band should be clearly resolved with minimal contaminating species.

Which isoform or processing form of Matriptase-2 does this recombinant represent — full-length or the serine protease domain only?

This recombinant corresponds to the extracellular serine protease domain of human Matriptase-2 (TMPRSS6), which harbors the catalytic activity relevant to substrate cleavage assays. The transmembrane anchor and cytoplasmic tail are excluded, consistent with how endogenous Matriptase-2 is shed from the cell surface in its active form. The catalytic triad (His, Asp, Ser) characteristic of S1-family trypsin-like serine proteases is intact. This soluble, active ectodomain format is directly relevant to hemojuvelin cleavage studies and inhibitor screening without requiring membrane reconstitution.

What substrate does Matriptase-2 cleave and how do I set up a fluorogenic activity assay?

Matriptase-2's best-characterized physiological substrate is membrane-bound hemojuvelin (HJV), which it cleaves to suppress hepcidin transcription. For in vitro activity assays, synthetic fluorogenic peptide substrates carrying a trypsin-like (Arg/Lys-P1) cleavage motif — such as Boc-Gln-Ala-Arg-AMC or Tosyl-Gly-Pro-Arg-AMC — are commonly used and work well with this recombinant. Assay the enzyme in 50 mM Tris-HCl pH 7.5, 150 mM NaCl (our storage buffer is directly compatible). A starting enzyme concentration of 10–50 nM with substrate at 100–200 µM gives a robust linear signal window in a 37°C fluorimetric read.

What buffer conditions are optimal for Matriptase-2 protease activity assays and inhibitor IC50 measurements?

The recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, which is directly compatible with standard serine protease activity assays — no buffer exchange is required for most setups. For IC50 measurements, dilute the enzyme into assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20 to minimize surface adsorption) and pre-incubate with inhibitor for 30 minutes at room temperature before substrate addition. Keep glycerol below 1% in the final assay volume to avoid inhibiting substrate turnover. PMSF and other serine protease inhibitors should be excluded from all controls.

What starting concentration of recombinant Matriptase-2 should I use for an inhibitor screening experiment?

For inhibitor potency measurements, we recommend titrating the enzyme between 5–50 nM in your assay and selecting a concentration that gives 30–50% substrate conversion at your chosen time point under Vmax-linear conditions — this ensures the assay is sensitive to both competitive and slow-binding inhibitors. Because Matriptase-2 activity can vary with lot-to-lot glycosylation differences, confirm the active enzyme concentration by active-site titration with a tight-binding standard inhibitor (e.g., PMSF or a peptide chloromethyl ketone) before committing to a full compound screen.

Can I use recombinant Matriptase-2 as a positive control for Western blot with the matched TPB antibody RP-Matriptase2?

Yes — this is one of the primary intended uses of REC-Matriptase2 alongside RP-Matriptase2 (/anti-matriptase-2-rabbit-polyclonal-antibody). The recombinant and the antibody are validated in the same lab, so band identity is unambiguous: expect a signal at 95–110 kDa on a reducing gel. Load 20–50 ng of recombinant per lane for a clean, non-saturating signal with standard ECL detection. This combination is particularly useful for confirming antibody lot-to-lot consistency and for spiking into cell lysates when endogenous Matriptase-2 expression is low, as is common in many non-hepatic cell lines.

How much recombinant Matriptase-2 should I load for Western blot positive control and what band should I expect?

Load 20–50 ng per lane when using RP-Matriptase2 (SKU: RP-Matriptase2) under standard ECL chemiluminescence conditions. The dominant band will appear between 95–110 kDa on a reducing SDS-PAGE gel, reflecting glycosylation from the HEK293 expression system. If your lysate of interest is from cells with high endogenous Matriptase-2 (e.g., liver-derived lines), consider loading 10–20 ng to keep the recombinant control from overshadowing the endogenous signal. Under non-reducing conditions, migration may shift slightly depending on disulfide bond content in the extracellular domain.

How should I store and handle recombinant Matriptase-2 to maintain activity, and how many freeze-thaw cycles are acceptable?

Store at -20°C in the single-use aliquots provided. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is formulated to protect activity during frozen storage; the glycerol serves as a cryoprotectant. Avoid repeated freeze-thaw cycles — we recommend no more than one additional freeze-thaw beyond initial thaw. Once thawed, keep the protein on ice and use within 24 hours. For experiments requiring serial dilutions, prepare working stocks in assay buffer containing 0.1% BSA to prevent surface adsorption losses at low nanomolar concentrations. Do not dilute into water alone.