TMPRSS-2 (Recombinant)

Frequently Asked Questions

What molecular weight band does recombinant TMPRSS-2 run at on SDS-PAGE or Western blot?

The full-length TMPRSS-2 precursor (492 aa, UniProt O15393) has a predicted molecular weight of ~54 kDa, but glycosylation in HEK293 cells typically shifts the apparent MW to ~60–70 kDa under reducing SDS-PAGE conditions. After autocatalytic zymogen processing, the catalytic chain runs at approximately 30–35 kDa. When running REC-TMPRSS2 as a Western blot standard, expect a predominant band in the 60–70 kDa range for the unprocessed form, with a secondary band at ~30–35 kDa reflecting processed enzyme. Purity is >95% by SDS-PAGE.

Is REC-TMPRSS2 the full-length precursor or the processed catalytic chain, and does that affect activity?

REC-TMPRSS2 is produced as the full-length 492 aa precursor in HEK293 cells. Consistent with native TMPRSS-2 biology, autocatalytic cleavage at an Arg residue liberates the trypsin-like serine protease catalytic chain carrying the canonical His-Asp-Ser triad. Because the protein is expressed in a mammalian system, zymogen processing occurs during production, so the preparation contains a mixture of precursor and active catalytic chain. For activity-dependent experiments — peptide cleavage assays, inhibitor screens — the active cleaved form is the relevant species and is functional as supplied.

What fluorogenic peptide substrate should I use for a TMPRSS-2 activity assay with this recombinant?

Boc-Gln-Ala-Arg-AMC is the most widely cited fluorogenic substrate for TMPRSS-2 in the published literature, reflecting the enzyme's preference for cleavage after basic (Arg/Lys) P1 residues. Other Arg-P1 AMC substrates (e.g., Boc-Phe-Ser-Arg-AMC) are also compatible. For a standard activity assay, use 100–500 µM substrate in 50 mM Tris-HCl pH 8.0, 150 mM NaCl at 37°C, monitoring AMC release at Ex/Em 355/460 nm. Initial velocity should be linear over at least 30 minutes before substrate depletion becomes significant.

What buffer and pH conditions are optimal for TMPRSS-2 serine protease activity assays?

TMPRSS-2 is a trypsin-like serine protease with maximum activity in the pH 7.5–8.5 range. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible with direct dilution into assay buffer. For activity assays, we recommend 50 mM Tris-HCl pH 8.0, 150 mM NaCl, without added calcium (TMPRSS-2 is not a metalloproteinase). Keep glycerol ≤2% in the final assay volume to avoid viscosity artifacts. Pre-warm enzyme and substrate to 37°C separately before mixing to obtain reproducible initial-rate measurements.

What starting enzyme concentration and inhibitor controls should I use for a TMPRSS-2 IC50 screen?

For IC50 determinations, 1–5 nM REC-TMPRSS2 is a reasonable starting point with 100–200 µM Boc-Gln-Ala-Arg-AMC substrate (below or at Km). Published IC50 values for common comparators — camostat mesylate (~6 nM), nafamostat (~0.3 nM), and aprotinin (~100 nM) — provide useful bracket controls to validate assay window and Z-factor. Pre-incubate enzyme with inhibitor for 15–30 minutes at room temperature before adding substrate. At these enzyme concentrations, Morrison tight-binding kinetics may apply for nanomolar inhibitors; adjust analysis accordingly.

Can I use REC-TMPRSS2 as a positive control for Western blot with the matched anti-TMPRSS-2 antibody RP-TMPRSS2?

Yes, REC-TMPRSS2 is the validated positive control for the matched rabbit polyclonal antibody RP-TMPRSS2 (/anti-tmprss-2-rabbit-polyclonal-antibody). The two are produced in the same laboratory and verified together. For a clean positive-control lane, load 50–100 ng of REC-TMPRSS2 alongside your cell lysate lanes. Expect the primary band at ~60–70 kDa (glycosylated precursor) and a secondary band at ~30–35 kDa. Because glycosylation patterns in the recombinant and endogenous protein are both derived from mammalian expression, band migration in cell lysates should closely match the recombinant standard.

How much REC-TMPRSS2 protein should I load to validate antibody RP-TMPRSS2 by Western blot?

For antibody validation purposes, a two-point loading series of 25 ng and 100 ng per lane is sufficient to demonstrate dose-dependent signal with RP-TMPRSS2 and to define the linear detection range. Run alongside a TMPRSS-2-positive cell lysate (e.g., Calu-3 or VCaP whole-cell lysate at 20–40 µg total protein). The recombinant lane confirms antibody specificity at the correct MW; the cell lysate lane confirms detection of endogenous protein. Purity of REC-TMPRSS2 is >95% by SDS-PAGE, so background bands in the recombinant lane are minimal.

How should I store and handle REC-TMPRSS2 to maintain serine protease activity over time?

REC-TMPRSS2 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. Avoid repeated freeze-thaw cycles; each additional cycle measurably reduces serine protease activity. Upon first thaw, briefly centrifuge (1,000 × g, 30 sec) and keep on ice during use. For dilutions below 10 µg/mL, add carrier protein (0.1% BSA, protease-free) to reduce adsorptive loss on tube surfaces. Aliquots are stable for ≥12 months at -20°C from the date of receipt when handled correctly.