TMPRSS-12 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant TMPRSS-12 on SDS-PAGE or Western blot?

The predicted molecular weight of TMPRSS-12 (Q86WS5) based on its 348-amino acid sequence is approximately 39 kDa. However, because this recombinant is produced in HEK293 mammalian cells, N-linked and O-linked glycosylation will shift the apparent molecular weight on SDS-PAGE to roughly 45–55 kDa under reducing conditions. The exact migration can vary slightly with gel percentage; we recommend a 10–12% polyacrylamide gel for clean resolution in this range. Under non-reducing conditions, expect a modestly higher apparent MW due to intramolecular disulfide architecture.

Is recombinant TMPRSS-12 full-length or a processed catalytic domain fragment?

This recombinant form (REC-TMPRSS12) encompasses the extracellular serine protease catalytic domain of TMPRSS-12, which is the enzymatically active region relevant to substrate cleavage and protein interaction studies. It does not include the single-pass transmembrane anchor or the short cytoplasmic tail present in the native type II transmembrane topology. This design yields a soluble, active protein suitable for biochemical assays, while still recapitulating authentic glycosylation patterns that prokaryotic or insect-cell systems would not provide.

What substrates does TMPRSS-12 cleave and which synthetic peptide substrate should I use in an activity assay?

TMPRSS-12 is a trypsin-like serine endopeptidase (EC 3.4.21.-) that cleaves after basic residues (Arg, Lys). For fluorogenic activity assays, Boc-Gln-Ala-Arg-AMC and Tos-Gly-Pro-Arg-AMC are commonly used substrates for type II transmembrane serine proteases in this family and are a reasonable starting point. In published biological contexts, TMPRSS-12 has been implicated in ADAM3 maturation on the sperm surface. Confirm substrate preference empirically with your specific lot by titrating substrate concentration from 50–500 µM and monitoring fluorescence release (Ex 360 nm / Em 460 nm) over 30–60 minutes at 37°C.

What assay buffer and conditions work best for TMPRSS-12 protease activity assays?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with direct use in activity assays and requires no buffer exchange for most applications. For optimal serine protease activity, a working assay buffer of 50 mM Tris-HCl pH 8.0, 100–150 mM NaCl, 0.01% Tween-20 is a good starting formulation. TMPRSS-12 is a serine protease, so avoid PMSF or other serine protease inhibitors in your assay. Conduct reactions at 37°C; BSA at 0.1 mg/mL can be added to stabilize the enzyme at low concentrations (< 50 nM).

What starting concentration of recombinant TMPRSS-12 should I use in a biochemical protease activity assay?

A practical starting point is 10–50 nM of REC-TMPRSS12 per reaction with a fluorogenic substrate at 100–200 µM. Titrate enzyme concentration to identify the linear range of product formation, typically within the first 15–30 minutes before substrate depletion. For inhibitor IC50 determinations, fix the enzyme at the lowest concentration giving a robust signal (often 10–20 nM) and titrate inhibitor across at least 8 concentrations spanning 3 log units. Ensure total DMSO from inhibitor stocks does not exceed 1% (v/v) in the final reaction volume to avoid serine protease inhibition artifacts.

Can I use REC-TMPRSS12 as a positive control for Western blot with the matched RP-TMPRSS12 rabbit polyclonal antibody?

Yes — this is one of the primary intended uses of REC-TMPRSS12 alongside RP-TMPRSS12 (/anti-tmprss-12-rabbit-polyclonal-antibody). The recombinant protein and the antibody are produced and validated in the same laboratory, ensuring antigen-antibody compatibility. Load 50–200 ng of REC-TMPRSS12 per lane on a reducing SDS-PAGE gel alongside your experimental lysates. You should detect a band at approximately 45–55 kDa under these conditions with RP-TMPRSS12 at a 1:500–1:2000 dilution. This positive control lane also allows you to confirm antibody specificity and lot-to-lot consistency in your hands.

How much recombinant TMPRSS-12 should I load for a Western blot positive control lane?

Load 50–200 ng per lane as a starting range when using RP-TMPRSS12 for detection. Begin at 100 ng if your chemiluminescence substrate is standard-sensitivity HRP; drop to 50 ng if using high-sensitivity ECL to avoid signal saturation. Run on a 10–12% SDS-PAGE gel under reducing conditions. The glycosylated recombinant will migrate at ~45–55 kDa, distinctly separated from most common housekeeping proteins. If you are establishing the assay for the first time, run a 3-point titration (50, 100, 200 ng) in parallel with your cell lysate to bracket the optimal loading amount.

How should I handle, dilute, and store recombinant TMPRSS-12 to preserve activity?

REC-TMPRSS12 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol as single-use aliquots. Store at -20°C upon receipt; avoid repeated freeze-thaw cycles, which progressively denature serine proteases. Thaw aliquots on ice and use within the same working session. For dilutions below 100 nM, use assay buffer supplemented with 0.1 mg/mL BSA as carrier to minimize adsorptive losses to tube walls. Purity is >90% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL, so no additional clean-up is needed for standard biochemical or cell-based assays.