Matriptase-3 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Matriptase-3 on SDS-PAGE or Western blot?

The full-length human Matriptase-3 (TMPRSS7, Q7RTY8) is 843 amino acids with a predicted unglycosylated MW of ~95 kDa. This recombinant is expressed in HEK293 cells, so N-linked glycosylation is expected to shift the apparent MW on SDS-PAGE to approximately 110–130 kDa under reducing conditions, depending on glycan occupancy. Under non-reducing conditions the band may run slightly differently due to intramolecular disulfide bonds. Always confirm band identity with a validated antibody such as RP-Matriptase3, which targets the serine protease domain.

What processing form does recombinant Matriptase-3 represent — full-length, zymogen, or activated catalytic domain?

This recombinant corresponds to the extracellular portion of Matriptase-3, encompassing the SEA, CUB, LDLRA, and serine protease domains, expressed without the N-terminal transmembrane anchor. It is produced as an active enzyme in HEK293 cells, meaning the serine protease domain is in its mature, processed conformation rather than as a zymogen. The active site catalytic triad (Ser, His, Asp) is intact. If your experiment requires a defined zymogen-to-active transition, we recommend confirming baseline amidolytic activity against a trypsin-like fluorogenic substrate before use.

What substrate does Matriptase-3 cleave and which fluorogenic peptide substrate should I use for an activity assay?

Matriptase-3 exhibits trypsin-like serine protease specificity, preferentially cleaving after Arg in the P1 position. For routine activity assays, the fluorogenic substrate Boc-Gln-Ala-Arg-AMC (or the widely used Boc-QAR-AMC) is appropriate. Alternatively, Tos-Gly-Pro-Arg-AMC (used for matriptase family members broadly) works well. Assay in 50 mM Tris-HCl pH 7.5, 150 mM NaCl at 37°C, monitoring fluorescence at Ex 380 nm / Em 460 nm. Run a no-enzyme blank in parallel. Serine protease inhibitors such as aprotinin or AEBSF serve as activity controls to confirm signal is Matriptase-3-specific.

What buffer conditions optimize Matriptase-3 recombinant protease activity assays, and does glycerol interfere?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with most activity assays when diluted at least 1:5 into assay buffer, reducing glycerol to ≤2%, a level that does not measurably inhibit serine proteases. Optimal activity is observed between pH 7.0–8.0; avoid going below pH 6.5 where the active-site histidine becomes protonated. Do not include EDTA (no metal cofactor requirement, but some commercial EDTA stocks carry trace serine protease inhibitor contaminants). DTT at >1 mM can reduce critical disulfide bonds and should be omitted.

What starting concentration of recombinant Matriptase-3 should I use for a fluorogenic protease activity assay?

A practical starting point is 10–50 nM Matriptase-3 with substrate concentrations ranging from 50–200 µM (ideally bracketing the Km, which for related matriptases is typically 100–400 µM for Arg-AMC substrates). Begin with 25 nM enzyme and 100 µM substrate in a 100 µL reaction volume at 37°C, measuring initial velocity over 30–60 minutes. Titrate enzyme concentration to keep substrate conversion below 15% to maintain initial-rate conditions. For IC50 determinations of inhibitors, fix enzyme at 20 nM and vary inhibitor concentration across at least eight points spanning three orders of magnitude.

Can I use Matriptase-3 recombinant protein as a positive control for Western blot with the RP-Matriptase3 rabbit polyclonal antibody?

Yes — this is one of the primary validated use cases. Load 50–100 ng of Matriptase-3 (Recombinant) per lane alongside your cell or tissue lysate samples. Run under reducing SDS-PAGE conditions; expect a band at approximately 110–130 kDa. The matched antibody RP-Matriptase3 (/anti-matriptase-3-rabbit-polyclonal-antibody) was raised and validated against this same recombinant-derived antigen in the same laboratory, so band identity is unambiguous. This pairing eliminates the common ambiguity of using a recombinant from one vendor to validate an antibody from another.

How much recombinant Matriptase-3 should I load as a Western blot positive control, and what band should I expect?

Load 50–150 ng per lane for a clean, non-saturating signal with the RP-Matriptase3 antibody at a 1:500–1:2000 dilution range (optimize per your detection system). On a 4–12% Bis-Tris gel under reducing conditions, expect the primary band at ~110–130 kDa due to HEK293-derived glycosylation. You may see a faint lower band (~95 kDa) representing partially deglycosylated or alternatively processed species — this is normal. If signal is weak, increase load to 200 ng before increasing antibody concentration, as excess antibody raises background on endogenous lysate lanes.

How should I store and handle recombinant Matriptase-3 to preserve protease activity, and what is the shelf life?

Store at -20°C in the single-use aliquots provided. Avoid repeated freeze-thaw cycles — each cycle can reduce serine protease activity by 10–20% due to aggregation of the hydrophobic protease domain. On ice, the protein is stable for 4–6 hours during a typical assay session. For working dilutions, prepare fresh in 50 mM Tris-HCl pH 7.5, 150 mM NaCl containing 0.1% BSA as a carrier to minimize adsorption to tube walls at low concentrations (<10 nM). When stored properly at -20°C with no freeze-thaw cycling, activity is stable for at least 12 months from the date of receipt.