TMPRSS-11-F (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant TMPRSS-11-F on SDS-PAGE or Western blot?

The full-length human TMPRSS-11-F protein is 438 amino acids, giving a theoretical MW of approximately 49 kDa. However, this HEK293-expressed recombinant carries native-like N-linked glycosylation, so the apparent MW on SDS-PAGE typically runs between 55–65 kDa under reducing conditions. Glycosylation heterogeneity can produce a diffuse or doublet band in this range — this is expected and does not indicate degradation. Under non-reducing conditions, disulfide-linked oligomers may appear at higher MW. Run the provided lot-specific SDS-PAGE image alongside your samples to confirm band position.

Is recombinant TMPRSS-11-F the full-length protein or a processed/cleaved form?

This recombinant corresponds to the extracellular serine protease domain of TMPRSS-11-F (UniProt Q6ZWK6), not the full-length type II transmembrane form. The transmembrane anchor and short cytoplasmic tail are absent, yielding a soluble, secreted format suitable for solution-phase assays. Like other TTSP family members, the catalytic domain can undergo autocatalytic or trans-proteolytic activation; the material supplied has been produced and quality-checked specifically for serine protease activity, confirming the active conformation is present. Lot-specific activity data are included with each shipment.

What substrates does TMPRSS-11-F cleave and which peptide substrate should I use for activity assays?

TMPRSS-11-F is a trypsin-like serine protease (EC 3.4.21.-) that cleaves after basic residues (Arg, Lys). Fluorogenic peptide substrates commonly used for HAT/DESC subfamily members include Boc-Gln-Ala-Arg-AMC and Tos-Gly-Pro-Arg-AMC (both available from commercial peptide suppliers). For initial activity confirmation, 100 µM Boc-Gln-Ala-Arg-AMC in assay buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% Tween-20) at 37°C with 50–200 ng recombinant enzyme per well (96-well format) is a practical starting point. Monitor AMC release at Ex/Em 380/460 nm.

What buffer conditions and pH are optimal for TMPRSS-11-F protease activity assays?

Serine proteases of the TTSP family, including TMPRSS-11-F, show peak trypsin-like activity between pH 7.5–8.5. A working assay buffer of 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% Tween-20 is recommended. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible with direct dilution into assay buffer; glycerol carried over at ≤1% (v/v) final concentration does not significantly affect AMC-based kinetic readouts. Avoid EDTA or DTT above 1 mM, as these can disrupt the catalytic serine–histidine–aspartate triad or reduce disulfide-dependent structural integrity.

What starting enzyme concentration should I use for TMPRSS-11-F inhibitor IC50 experiments?

For inhibitor dose-response assays, a starting enzyme concentration of 50–100 ng per well (approximately 1–2 nM in a 100 µL reaction, based on ~55 kDa apparent MW) is practical. This keeps substrate turnover in the linear range over a 30–60 minute window and avoids substrate depletion artifacts that compress IC50 estimates. Pre-incubate enzyme with inhibitor for 15–30 minutes at 25°C before initiating the reaction with substrate. Because TTSP activity can vary between lots, normalize enzyme input to RFU/min using a substrate-only control before comparing inhibitor data across experiments.

Can I use recombinant TMPRSS-11-F as a positive control for Western blot with the matched TPB antibody?

Yes — this recombinant is the recommended positive control for the matched rabbit polyclonal antibody RP-TMPRSS11F (/anti-tmprss-11f-rabbit-polyclonal-antibody), which was raised and validated in the same laboratory. The antibody has been validated on HEK293-expressed TMPRSS-11-F by Western blot, so band position, glycoform pattern, and signal intensity are directly comparable between recombinant control lanes and cell lysate lanes. Loading 20–50 ng of recombinant per lane under reducing denaturing conditions produces a clean, readily detectable band in the 55–65 kDa range with RP-TMPRSS11F at standard working dilutions.

How much recombinant TMPRSS-11-F should I load for a Western blot positive control lane?

Load 20–50 ng per lane for a standard chemiluminescent Western blot using RP-TMPRSS11F. At 20 ng, signal is typically sufficient with 1–2 hour ECL exposure on HRP-conjugated secondary; at 50 ng, the band is easily detectable even with shorter exposures or less sensitive film. Avoid loading above 100 ng — overloading can cause band smearing due to glycoform heterogeneity and may bleed into adjacent sample lanes. If using fluorescent secondaries or quantitative Western systems (e.g., LI-COR), 10–20 ng is usually adequate for a signal within the linear detection range.

How should I store and handle recombinant TMPRSS-11-F to preserve protease activity long-term?

Store at -20°C in single-use aliquots as supplied. Repeated freeze-thaw cycles progressively reduce serine protease activity and can promote aggregation; plan aliquot sizes around your typical per-experiment usage (e.g., 5–10 µg per tube). On the day of use, thaw on ice and keep at 4°C; activity is stable for up to 8 hours under these conditions. For dilutions below 10 µg/mL, add carrier protein (0.1% BSA, protease-free) to the working buffer to prevent adsorptive loss to tube walls. Do not vortex — gentle pipette mixing is sufficient.