TMPRSS-11-D (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant TMPRSS-11-D on SDS-PAGE or Western blot?

TMPRSS11D has a predicted molecular weight of approximately 46 kDa based on its 418-amino acid sequence. However, because this recombinant is produced in HEK293 cells, mammalian N-linked and O-linked glycosylation will shift the apparent molecular weight on SDS-PAGE — expect a broad or diffuse band in the 50–60 kDa range under denaturing, reducing conditions. If you observe a doublet, this likely reflects heterogeneous glycoforms of the processed catalytic chain. Comparing under reducing vs. non-reducing conditions can help resolve the autoproteolytically cleaved N- and C-terminal chains.

What form of TMPRSS-11-D does this recombinant represent — full-length, catalytic domain, or cleaved form?

TMPRSS11D undergoes autoproteolytic cleavage at the cell membrane to yield a non-catalytic N-terminal stem region and a catalytic C-terminal serine protease domain. The HEK293 expression system used here recapitulates the native secretory pathway, so this recombinant is expected to be present predominantly as the processed, active form containing the catalytic domain. This makes it directly relevant to studies of the mature enzyme in airway physiology, viral priming assays, or inhibitor profiling — rather than requiring you to activate an inactive zymogen in vitro.

What substrate should I use to measure TMPRSS-11-D protease activity in a fluorescence assay?

Boc-Phe-Ser-Arg-4-methylcoumaryl-7-amide (Boc-FSR-MCA) is the most thoroughly characterised synthetic substrate for TMPRSS11D (HAT) activity, reflecting the enzyme's strong preference for arginine at the P1 position. Cleavage releases the 7-amino-4-methylcoumarin (AMC) fluorophore, monitored at Ex 380 nm / Em 460 nm. Prepare substrate stocks in DMSO and dilute to 50–200 µM in assay buffer, keeping DMSO ≤1% v/v in the final reaction. A standard substrate concentration range of 50–500 µM will allow basic Michaelis-Menten kinetic characterisation.

What buffer conditions are optimal for a TMPRSS-11-D activity assay, and does the storage buffer interfere?

TMPRSS11D displays optimal activity at pH 8.6 in published literature. For fluorometric assays, use 50 mM Tris-HCl pH 8.6, 150 mM NaCl as the activity buffer; this differs from the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol). Dilute the enzyme at least 10-fold into activity buffer immediately before use to reduce glycerol to ≤1%, which can otherwise suppress activity at higher concentrations. Glycerol at 10% in the storage buffer is included for cryoprotection and does not compromise activity when properly diluted prior to the assay.

What starting enzyme concentration should I use for a TMPRSS-11-D inhibitor IC50 experiment?

As a starting point, 1–5 nM recombinant TMPRSS11D in activity buffer (50 mM Tris-HCl pH 8.6, 150 mM NaCl) paired with 100 µM Boc-FSR-MCA substrate is a reasonable assay window for inhibitor titrations. Keep enzyme concentration well below the Ki of the inhibitor series to ensure competitive binding conditions and avoid tight-binding artefacts. Pre-incubate enzyme with inhibitor for 15–30 minutes at 25°C before initiating the reaction with substrate. Confirm linearity of AMC release over the measurement window (typically 30–60 minutes) before committing to a full IC50 curve.

Can I use REC-TMPRSS11D as a positive control for Western blot with the matched TMPRSS-11-D antibody RP-TMPRSS11D?

Yes — REC-TMPRSS11D is specifically matched to antibody RP-TMPRSS11D (/anti-tmprss-11d-rabbit-polyclonal-antibody) and is validated as a positive control for Western blot. Load 50–100 ng of recombinant per lane under reducing, denaturing conditions alongside your cell lysate samples. The antibody recognises the processed catalytic form, so the recombinant band at ~50–60 kDa serves as a reliable size reference. Because both the antibody and recombinant originate from the same production programme, this pairing is also well-suited for antibody validation experiments demonstrating on-target specificity.

How much recombinant TMPRSS-11-D should I load for Western blot to get a clean positive control band?

50–100 ng per lane is the recommended loading range for a clean, well-resolved positive control band with RP-TMPRSS11D antibody. At 50 ng you should achieve a strong signal without smearing; if background is an issue in your lysate lanes, dropping to 25 ng and extending antibody incubation time is preferable to reducing primary antibody concentration. Run the recombinant in a dedicated lane flanking your experimental samples rather than mixing it into the same well, as the glycosylated band can broaden and overlap adjacent lanes at higher loads.

How should I handle, dilute, and store recombinant TMPRSS-11-D to preserve activity over time?

REC-TMPRSS11D is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and should be stored at -20°C in single-use aliquots. Repeated freeze-thaw cycles cause measurable activity loss — aliquot to your typical working volume (e.g., 5–10 µg) before first use. On the day of the experiment, thaw on ice and dilute directly into pre-warmed assay buffer; avoid holding diluted enzyme at room temperature for more than 1 hour. At -20°C in the original buffer, activity is stable for at least 12 months. Do not store at 4°C long-term, as serine proteases are susceptible to autoproteolytic degradation at non-frozen temperatures.