Enteropeptidase (Recombinant)

Frequently Asked Questions

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

The catalytic light chain of human enteropeptidase — the enzymatically active serine protease domain — runs at approximately 35–40 kDa under reducing SDS-PAGE conditions. The full-length protein (1,019 aa, UniProt P98073) has a predicted MW of ~116 kDa, but the recombinant is produced as the processed light chain fragment rather than the intact single-pass transmembrane form. Because the protein is expressed in HEK293 cells and carries N-linked glycosylation, the apparent MW on gel typically runs slightly higher than the sequence-predicted value. Confirm identity with our matched antibody RP-Enteropeptidase.

What processing form is the Triple Point Biologics recombinant Enteropeptidase — full-length or catalytic light chain?

This recombinant corresponds to the catalytic light chain of human enteropeptidase (EC 3.4.21.9), which carries the serine protease domain responsible for substrate cleavage. The full-length TMPRSS15 protein is a type II transmembrane protease; the light chain is the soluble, enzymatically active fragment released after endogenous processing. Expression in HEK293 cells supports disulfide bond formation critical for proper folding of this domain. Researchers planning structural or binding studies requiring the full ectodomain, including the heavy chain, should note that this product is not the full-length transmembrane form.

What substrate does recombinant Enteropeptidase cleave and what sequence should I use for activity assays?

Enteropeptidase cleaves with high specificity after the pentapeptide sequence Asp-Asp-Asp-Asp-Lys (DDDDK↓), making Gly-Asp-Asp-Asp-Asp-Lys-β-naphthylamide (Gly-(Asp)4-Lys-βNA) the standard fluorogenic substrate for activity assays. For cleavage of trypsinogen, the natural substrate, the enzyme recognizes the DDDDK↓Ile junction in the activation peptide. Synthetic DDDDK-containing peptide substrates or fusion-tag removal applications (e.g., releasing His-tagged proteins after DDDDK cleavage) are the most common lab uses. Monitor fluorogenic substrate cleavage at excitation ~335 nm / emission ~410 nm for a straightforward continuous activity readout.

What buffer conditions work best for Enteropeptidase activity assays, and does the storage buffer interfere?

Enteropeptidase activity assays are routinely run in 50 mM Tris-HCl pH 7.5–8.0, 150 mM NaCl, with Ca²⁺ ions (1–5 mM CaCl₂) often included to support serine protease stability. The storage buffer for this recombinant — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is fully compatible; glycerol at ≤1% final concentration in the reaction does not significantly inhibit activity at typical working dilutions. Avoid reducing agents such as DTT or β-mercaptoethanol above 1 mM, as they can disrupt the disulfide bonds required for the light chain's active conformation. pH optimum is 7.5–8.0.

What starting concentration of recombinant Enteropeptidase should I use for a trypsinogen activation or substrate cleavage assay?

For fluorogenic peptide substrate assays (e.g., Gly-(Asp)4-Lys-βNA), a starting concentration of 1–10 nM recombinant Enteropeptidase in a 50–100 µL reaction is a reasonable titration range. For trypsinogen activation assays, 0.1–1 µg enzyme per 10–50 µg trypsinogen is a common starting ratio; optimize based on your trypsinogen source and desired activation kinetics. Because specific activity can vary with storage history, always run a substrate titration on the first use of a new aliquot. Spin briefly at 10,000 × g before opening to collect any condensate from freeze-thaw.

Can I use recombinant Enteropeptidase as a positive control for Western blot with the RP-Enteropeptidase antibody?

Yes — this recombinant is the most direct positive control for the matched RP-Enteropeptidase rabbit polyclonal antibody (SKU: RP-Enteropeptidase). Because both products come from the same TPB workflow, antigen–antibody compatibility is confirmed. Load 50–200 ng of recombinant per lane under reducing conditions; this should yield a clean band at ~35–40 kDa (glycosylated light chain) detectable with a standard HRP-secondary at 1:5,000–1:10,000 dilution. This loading range avoids oversaturation while remaining well above the antibody's detection threshold, giving you a reliable size reference alongside endogenous samples from duodenal lysates.

How much recombinant Enteropeptidase should I load as a positive control and what band will RP-Enteropeptidase antibody detect?

Load 100–200 ng per lane for a robust positive control signal. The RP-Enteropeptidase antibody will detect the catalytic light chain at ~35–40 kDa (apparent MW under reducing SDS-PAGE, shifted slightly above sequence-predicted due to glycosylation from HEK293 expression). If you are also running duodenal epithelial cell lysate alongside, the same band should align. For lower-abundance samples where you need a faint positive control to avoid obscuring nearby bands, 50 ng is generally still detectable. Transfer efficiency for this MW range is reliable on standard PVDF at 100 V/1 h; no special transfer conditions are required.

How should I store recombinant Enteropeptidase and what is its shelf life after thawing?

Store at -20°C in single-use aliquots as supplied. The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is formulated to maintain stability through long-term frozen storage, with shelf life of at least 12 months from the date of manufacture when stored correctly. Once thawed, keep on ice and use within the same working session; do not refreeze. Repeated freeze-thaw cycles cause measurable loss of specific activity for serine proteases and should be avoided. If your experiment requires multiple uses, aliquot to smaller volumes upon first receipt before freezing. Do not store at 4°C long-term.