TMPRSS-13 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant TMPRSS-13 on SDS-PAGE or Western blot?

The full-length human TMPRSS-13 protein (UniProt Q9BYE2) has a predicted molecular weight of ~66 kDa based on its 586-amino-acid sequence. Due to N-linked glycosylation introduced during HEK293 expression — which mirrors the native glycosylation pattern of the TTSP ectodomain — the apparent MW on reducing SDS-PAGE typically runs between 70–80 kDa. If you are running non-reducing conditions, disulfide-stabilized conformers may shift the band further. Always compare against a prestained ladder calibrated in the 50–100 kDa range.

What processing or isoform state is this TMPRSS-13 recombinant — zymogen or activated form?

This recombinant is produced as the catalytically active ectodomain of TMPRSS-13, encompassing the serine protease domain. TMPRSS-13 belongs to the mosaic serine protease subgroup of the TTSP family and undergoes autocatalytic or trans-activation cleavage at a conserved Arg/Lys site in the activation loop. The HEK293 expression system supports the disulfide bonding required for proper domain folding; however, the precise zymogen-to-active ratio in the final lot is confirmed by activity assay before release. A certificate of activity is available on request.

What substrates does TMPRSS-13 cleave and which fluorogenic peptide substrate should I use in a activity assay?

TMPRSS-13 documented biological substrates include the proform of PRSS8/prostasin and the proform of HGF — both cleaved at basic residue motifs. For in vitro fluorogenic assays, Boc-Gln-Ala-Arg-AMC and Tos-Gly-Pro-Arg-AMC are suitable trypsin-like serine protease substrates and have been used with closely related TTSPs. Start at 100–200 µM substrate concentration with 0.5–2 µg/mL recombinant TMPRSS-13 in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl). Monitor AMC release at Ex 360 nm / Em 460 nm. Titrate enzyme concentration to keep initial velocity in the linear range.

What is the recommended assay buffer and pH for TMPRSS-13 protease activity in vitro?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — serves as a practical starting point for activity assays. TMPRSS-13 is a serine protease with preference for basic residues (Arg/Lys at P1), and its activity is generally optimal near physiological pH (7.4–8.0). Avoid EDTA, which is not required for this serine protease but is non-inhibitory; standard serine protease inhibitors (PMSF, AEBSF, benzamidine) must be excluded from assay buffers. BSA (0.01–0.1%) can be added to reduce non-specific surface adsorption in low-enzyme-concentration experiments.

How do I determine a starting concentration of TMPRSS-13 recombinant for an IC50 inhibitor screening experiment?

For IC50 measurements, the enzyme concentration should be well below the Ki of the tightest inhibitor you intend to rank — typically 0.5–2 nM total active enzyme — to avoid tight-binding artefacts (Cheng-Prusoff applies). Convert the supplied protein concentration (µg/mL) to molar using the apparent MW of ~70–75 kDa. Run a substrate titration (Km determination) first using the Boc-Gln-Ala-Arg-AMC or equivalent substrate, then set substrate at ~Km for IC50 experiments. This ensures the measured IC50 is easily corrected to Ki using the standard competitive inhibition equation.

Can I use REC-TMPRSS13 as a positive control antigen on Western blot with the matched TPB antibody RP-TMPRSS13?

Yes — this is one of the primary intended uses of the pairing. REC-TMPRSS13 and the rabbit polyclonal antibody RP-TMPRSS13 are produced and validated in the same laboratory, guaranteeing epitope compatibility. Load 20–50 ng of REC-TMPRSS13 per lane on a 10% SDS-PAGE gel under reducing conditions; RP-TMPRSS13 should detect the ~70–80 kDa glycosylated band cleanly. This positive control is especially useful when validating antibody lot-to-lot consistency or when setting up TMPRSS-13 detection in a new cell lysate panel. See the RP-TMPRSS13 product page (/anti-tmprss-13-rabbit-polyclonal-antibody) for recommended dilutions.

How much recombinant TMPRSS-13 should I load as a Western blot positive control to get a clean band?

20–50 ng per lane is the recommended starting range when using RP-TMPRSS13 at standard working dilutions. At 20 ng you should obtain a detectable band without saturating the signal; 50 ng provides a robust reference band suitable for molecular weight confirmation alongside cell lysates. Use reducing SDS-PAGE sample buffer; boiling for 5 min at 95°C is appropriate. The glycosylated band will appear at approximately 70–80 kDa. If running alongside high-protein-content tissue lysates, load the recombinant in a dedicated lane rather than mixing, to avoid signal interference.

How should I store and handle REC-TMPRSS13 to preserve enzymatic activity — can I refreeze unused protein?

REC-TMPRSS13 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol in single-use aliquots and should be stored at -20°C. Repeated freeze-thaw cycles measurably reduce serine protease activity — even one additional cycle can result in 20–30% activity loss in our experience with TTSP-family ectodomains. Thaw each aliquot on ice, use what you need, and discard the remainder rather than refreezing. If you anticipate multiple uses within a week, a working aliquot may be kept at 4°C for up to 5–7 days; add BSA to 0.1 mg/mL to stabilize dilute preparations.