Tripeptidyl Peptidase-2 (Recombinant)

Frequently Asked Questions

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

The full-length human TPP2 protein (UniProt P29144) is 1,249 amino acids, predicting an unmodified MW of ~138 kDa. HEK293-expressed material carries mammalian glycosylation, so the observed band on reducing SDS-PAGE typically migrates at approximately 140–145 kDa. Under non-denaturing conditions native TPP2 forms large oligomeric complexes (>1 MDa), so non-reducing or native PAGE will give a very different picture. For routine SDS-PAGE positive-control lanes, load under fully denaturing, reducing conditions and expect a single band in the 140–145 kDa range, confirmed at >90% purity by Coomassie or silver stain.

Is this recombinant TPP2 the full-length protein or a catalytic fragment, and does isoform matter for my assay?

This preparation is full-length human TPP2 (residues 1–1,249), expressed in HEK293 cells. A shorter splice variant (sometimes called TPP2-short) exists but lacks physiological relevance in most cytosolic degradation studies. For fluorogenic exopeptidase assays measuring sequential tripeptide release, full-length material is essential because oligomeric assembly — dependent on the C-terminal domain — is required for full catalytic efficiency. If your assay involves monitoring complex formation by native PAGE or size-exclusion chromatography, confirm oligomerization is intact prior to kinetic measurements; the HEK293 expression system is chosen specifically to support correct folding and assembly.

What substrates does recombinant Tripeptidyl Peptidase-2 cleave and which fluorogenic substrate is recommended for activity assays?

TPP2 is a subtilisin-type serine exopeptidase (EC 3.4.14.10) that releases N-terminal tripeptides from oligopeptide substrates. The most widely used fluorogenic substrate for in vitro activity assays is Ala-Ala-Phe-7-amido-4-methylcoumarin (AAF-AMC), which TPP2 cleaves to release the fluorescent AMC leaving group (Ex 360 nm / Em 460 nm). Substrate concentration should be titrated around a reported Km of ~0.1–0.3 mM for AAF-AMC. GluGluPhe-AMC is an alternative for selectivity profiling. Ensure substrate stocks are prepared in DMSO (<1% final v/v) to avoid activity interference.

What buffer conditions are optimal for a recombinant TPP2 fluorogenic activity assay?

Recombinant TPP2 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and is active in this buffer. For fluorogenic assays, dilute into assay buffer of 50 mM Tris-HCl pH 7.5 or 50 mM HEPES pH 7.4, 100–150 mM NaCl. TPP2 is a serine protease — include 1 mM DTT to maintain reducing conditions and prevent non-specific oxidative inactivation, but avoid metal chelators (EDTA) in the assay well unless specifically testing inhibitor mechanism. Reactions run at 37°C with 30–60 min pre-incubation of enzyme before substrate addition generally yield the most reproducible Vmax measurements.

What starting enzyme concentration should I use when setting up a TPP2 inhibitor IC50 assay?

For a standard AAF-AMC fluorogenic IC50 assay in 96- or 384-well format, start with 2–10 nM recombinant TPP2 per well, titrating inhibitor across 8–10 concentrations spanning at least 3 log units around the anticipated IC50. Confirm that fluorescence signal is linear with enzyme concentration and that substrate turnover remains below 10% at the assay endpoint to satisfy steady-state assumptions. Protein concentration of the supplied stock should be verified by absorbance at A280 or BCA assay before use, as 10% glycerol in the storage buffer can affect some colorimetric methods — account for this when calculating dilutions.

Can I use this recombinant TPP2 as a Western blot positive control with the matched Triple Point Biologics antibody?

Yes — this recombinant is the validated positive-control antigen for the Triple Point Biologics matched rabbit polyclonal antibody (SKU: RP-Tripeptidyl Peptidase2; see /anti-tripeptidyl-peptidase2-rabbit-polyclonal-antibody). The two products are produced in the same laboratory, and compatibility for Western blot has been confirmed. Load 10–50 ng of recombinant TPP2 per lane under reducing SDS-PAGE conditions alongside your cell lysate samples. A clean band at 140–145 kDa in the recombinant lane directly confirms antibody performance and validates any band of equivalent MW observed in experimental lanes.

How much recombinant TPP2 should I load for a Western blot positive control lane?

Load 20–50 ng per lane as a starting point when pairing with the matched RP-Tripeptidyl Peptidase2 rabbit polyclonal antibody (SKU: RP-Tripeptidyl Peptidase2). At this range, a distinct band at ~140–145 kDa is detectable with standard chemiluminescent ECL without saturating the detector, allowing direct comparison to endogenous TPP2 signal in adjacent lysate lanes. If you are running a lower-sensitivity detection system, scale up to 100 ng. Dilute the recombinant stock in 1× Laemmli buffer immediately before use; do not store diluted material, as repeated dilution from frozen aliquots accelerates activity loss.

How should I store and handle recombinant TPP2 to maintain activity, and what is the shelf life?

Store at -20°C in single-use aliquots immediately upon receipt. The supplied buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is formulated for long-term frozen storage; the glycerol component minimizes freeze-thaw damage. Avoid repeated freeze-thaw cycles: each cycle can reduce measurable exopeptidase activity by 15–30% in our internal validation data. Shelf life is 12 months from date of manufacture when stored correctly. For working dilutions prepared on the day of assay, keep on ice and use within 4–6 hours. Endotoxin is <0.1 EU/µg by LAL, so no additional depyrogenation step is required for cell-based assays.