TMPRSS-4 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant TMPRSS-4 on SDS-PAGE or Western blot?

The full-length human TMPRSS-4 (UniProt Q9NRS4) has a predicted molecular weight of ~48 kDa from its 437-amino-acid sequence, but the HEK293-expressed recombinant typically migrates at ~55–60 kDa under denaturing conditions due to mammalian N-linked glycosylation. Under reducing versus non-reducing conditions the apparent MW should not shift dramatically, as the active catalytic fragment is generated by cleavage of the zymogen and may appear as a doublet near 30–35 kDa in preparations enriched for the processed form. Confirm your observed band using matched antibody RP-TMPRSS4 as a direct reference.

What processing form is the recombinant TMPRSS-4 supplied in — zymogen or active cleaved form?

REC-TMPRSS4 is supplied as the active enzyme. During HEK293 expression, TMPRSS4 undergoes autocatalytic or trans-proteolytic cleavage of its zymogen precursor, releasing the extracellular serine protease domain. The recombinant is produced and quality-controlled as this catalytically competent form rather than the uncleaved single-chain precursor. If your experimental model specifically requires the zymogen (e.g., to study activation kinetics), note that the supplied material will not reflect that unprocessed state and you should account for this in assay design.

What substrate should I use to measure TMPRSS-4 serine protease activity in a fluorescent activity assay?

TMPRSS4 is a trypsin-like serine protease that cleaves after basic residues (Arg, Lys). The most commonly used fluorogenic substrates are Boc-Gln-Ala-Arg-AMC and Tos-Gly-Pro-Arg-AMC (excitation ~380 nm, emission ~460 nm). For a standard kinetic assay, prepare 50–200 µM substrate in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20) and use 0.5–2 µg/mL REC-TMPRSS4. Monitor fluorescence continuously at 37°C. Include a serine protease inhibitor control (e.g., PMSF or aprotinin) to confirm signal specificity.

What buffer conditions are optimal for TMPRSS-4 recombinant enzyme activity and stability?

REC-TMPRSS4 is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and is fully compatible with assay buffers in the pH 7.0–8.0 range. Avoid EDTA or chelating agents in activity assays, as some serine proteases are sensitive to metal depletion; however, TMPRSS4 is not a metalloprotease and does not require supplemental CaCl₂. Reducing agents such as DTT above 1 mM can compromise disulfide-dependent active-site geometry — keep them at ≤0.5 mM if required. For functional downstream assays involving pro-uPA cleavage, physiological pH (7.4) in HEPES- or Tris-buffered saline is appropriate.

What starting concentration of recombinant TMPRSS-4 should I use to cleave pro-uPA (PLAU) substrate in vitro?

For cleavage of recombinant pro-uPA (PLAU) into its active two-chain form, a reasonable starting point is 0.5–2 µg of REC-TMPRSS4 per 10 µg of pro-uPA substrate, incubated at 37°C for 60–120 minutes in 50 mM Tris-HCl pH 7.5, 150 mM NaCl. Resolve the reaction by non-reducing SDS-PAGE and probe with an anti-uPA antibody to confirm the ~54 kDa single-chain to ~33 kDa + ~18 kDa two-chain conversion. Titrate enzyme-to-substrate ratio empirically if your pro-uPA source differs in glycosylation or specific activity from recombinant standards.

Can I use recombinant TMPRSS-4 as a Western blot positive control for the RP-TMPRSS4 antibody?

Yes — REC-TMPRSS4 and RP-TMPRSS4 are produced and validated together in the same laboratory specifically for this purpose. Load 50–100 ng of REC-TMPRSS4 per lane alongside your cell or tissue lysate. The rabbit polyclonal RP-TMPRSS4 (see /anti-tmprss-4-rabbit-polyclonal-antibody) is validated for Western blot and should produce a clean band at ~55–60 kDa (glycosylated recombinant) that serves as an unambiguous size marker and signal-intensity reference. This pairing is particularly useful when validating the antibody in a new cell line or tissue context where endogenous TMPRSS4 expression is uncertain.

How much recombinant TMPRSS-4 should I load for a Western blot positive control lane?

50–100 ng per lane is sufficient for a strong, clean signal with RP-TMPRSS4 at a 1:1,000–1:2,000 primary antibody dilution, standard HRP-conjugated secondary, and ECL detection. At 50 ng you will see a clearly detectable band without overwhelming adjacent sample lanes; at 100 ng the band is robust enough to serve as a reliable reference even with shorter exposure times. Because REC-TMPRSS4 is supplied at >90% purity by SDS-PAGE with endotoxin <0.1 EU/µg, background from contaminants is minimal and will not confound band interpretation.

How should I handle and store recombinant TMPRSS-4 to preserve enzymatic activity over time?

REC-TMPRSS4 is shipped on dry ice and should be stored at -20°C immediately upon receipt. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) stabilizes the enzyme through freeze-thaw; however, repeated freeze-thaw cycles progressively reduce specific activity — aliquot into single-use volumes before freezing. On the day of use, thaw on ice and dilute into pre-warmed assay buffer immediately before the experiment. Do not store diluted enzyme at 4°C for more than 24 hours. For long-term storage beyond 6 months, verify activity with a fluorogenic substrate benchmark before committing to a critical experiment.