TMPRSS-5 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant TMPRSS-5 on SDS-PAGE or Western blot?

The full-length human TMPRSS-5 protein is 457 amino acids, giving a predicted unmodified MW of ~51 kDa. However, because this recombinant is expressed in HEK293 cells, N-linked glycosylation adds appreciable mass — expect a diffuse band running at approximately 55–65 kDa under denaturing, reducing conditions. The smearing is characteristic of heterogeneous glycosylation and does not indicate degradation. If you treat the protein with PNGase F prior to SDS-PAGE, the band sharpens and migrates closer to the predicted 51 kDa backbone, which can be a useful glycosylation control in your experiment.

Is recombinant TMPRSS-5 full-length or a processed/truncated fragment, and does it include the transmembrane domain?

The recombinant TMPRSS-5 supplied here corresponds to the extracellular protease-competent region of the protein and does not include the N-terminal transmembrane anchor. Retaining the transmembrane domain in a soluble recombinant format would result in aggregation-prone material incompatible with standard solution-phase assays. The expressed fragment encompasses the serine protease domain and associated SRCR and low-density lipoprotein receptor domains, preserving the active-site triad (His, Asp, Ser) geometry required for trypsin-like catalytic activity. This is the form relevant to most substrate cleavage and inhibitor studies.

What substrates can I use to measure TMPRSS-5 serine protease activity in a fluorescence assay?

TMPRSS-5 is a trypsin-like serine protease (EC 3.4.21.-) and cleaves after basic residues (Arg, Lys). The fluorogenic substrate Boc-Gln-Ala-Arg-AMC (or the simpler Boc-Val-Pro-Arg-AMC) is a practical starting point at 50–200 µM in assay buffer. Tos-Gly-Pro-Arg-AMC, widely used for other TMPRSS family members, is also compatible. Monitor AMC release at excitation 380 nm / emission 460 nm. Run a no-enzyme blank and a heat-inactivated enzyme control in parallel to confirm signal is protease-dependent rather than substrate autofluorescence.

What buffer conditions and pH are optimal for a TMPRSS-5 activity assay?

TMPRSS-5 is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — a buffer compatible with direct use in activity assays, though glycerol carryover above ~2% can reduce some fluorogenic substrate signals. For kinetic assays, dilute into 50 mM HEPES pH 7.5, 150 mM NaCl, 0.01% Tween-20 (to limit surface adsorption). The protease shows trypsin-like activity across pH 7.0–8.0; avoid pH below 6.5 or above 9.0. Do not include EDTA — while TMPRSS-5 is a serine protease rather than a metalloprotease, chelators can destabilise the protein at low concentrations.

What starting concentration of recombinant TMPRSS-5 should I use for inhibitor IC50 measurements?

For IC50 determinations, titrate the enzyme first to establish a concentration that gives a linear signal over your assay window — typically 1–10 nM recombinant TMPRSS-5 with 100 µM fluorogenic substrate is a workable starting range. Keep enzyme concentration well below the Ki of your inhibitor to satisfy tight-binding assumptions (Morrison equation) if necessary. Run the inhibitor dilution series in triplicate with at least 30 min pre-incubation at room temperature before initiating with substrate. Confirm DMSO vehicle tolerance up to 1% v/v, as higher concentrations can measurably reduce serine protease activity.

How much recombinant TMPRSS-5 should I load as a Western blot positive control when validating the RP-TMPRSS5 antibody?

For Western blot positive control alongside anti-TMPRSS-5 rabbit polyclonal antibody (RP-TMPRSS5), load 50–100 ng of recombinant TMPRSS-5 per lane on a 10–12% SDS-PAGE gel. At this loading, RP-TMPRSS5 at a 1:500–1:2,000 primary antibody dilution (HRP-conjugated anti-rabbit secondary, 1:5,000–1:10,000) reliably produces a band in the 55–65 kDa region. The recombinant and RP-TMPRSS5 antibody are produced in the same laboratory from matched immunogen material, so this pairing provides a confirmed positive signal without ambiguity about epitope accessibility.

Can recombinant TMPRSS-5 be used to validate the RP-TMPRSS5 antibody for IHC or confirm antibody specificity in cell lysates?

Yes. Because REC-TMPRSS5 and RP-TMPRSS5 are produced as a matched pair, the recombinant is the most direct tool for antibody specificity confirmation. For cell lysate spike-in experiments, add 100–250 ng of recombinant TMPRSS-5 to your negative-control lysate (e.g., a TMPRSS5-knockout or low-expression cell line) and run alongside unspiked lysate — RP-TMPRSS5 should show a concentration-dependent signal only in the spiked lane. For IHC antigen retrieval validation, the recombinant can be spotted on nitrocellulose as a dot-blot control. See the RP-TMPRSS5 product page at /anti-tmprss-5-rabbit-polyclonal-antibody for validated dilution ranges.

How should I store and handle recombinant TMPRSS-5 to preserve enzymatic activity after receipt?

Upon receipt, briefly centrifuge the tube to consolidate the liquid, then aliquot into single-use volumes appropriate for your experiment before storing at -20°C. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) provides cryoprotection, but repeated freeze-thaw cycles measurably degrade serine protease activity — even two cycles can reduce specific activity by 20–40% in our internal QC monitoring. For experiments requiring several weeks of use, store working aliquots at 4°C for up to 7 days. Avoid diluting the stock below ~0.1 mg/mL without adding carrier protein (0.1% BSA) to limit adsorptive loss on tube surfaces.