DPP-4 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant DPP-4 run at on SDS-PAGE and Western blot?

The DPP-4 extracellular catalytic domain produced in HEK293 cells runs at approximately 90–110 kDa under reducing SDS-PAGE conditions. The theoretical MW of the 766-aa full-length human protein is ~88 kDa, but N-linked glycosylation added in the mammalian HEK293 expression system causes the characteristic upward shift. Expect a broad or slightly diffuse band reflecting heterogeneous glycoforms — this is normal and mirrors the native circulating enzyme. Under non-reducing conditions, the apparent MW is similar; no major disulfide-linked oligomers are observed at >95% purity by SDS-PAGE.

What processing form or isoform is the Triple Point Biologics recombinant DPP-4 — full-length or ectodomain?

REC-DPP4 corresponds to the soluble extracellular ectodomain of human DPP-4 (UniProt P27487), encompassing the catalytic domain responsible for dipeptidyl peptidase activity. The N-terminal transmembrane anchor is absent, yielding a secreted, soluble active enzyme — the same form found in circulation as soluble plasma DPP-4. This construct is therefore directly relevant to studies of the circulating enzyme pool, incretin hormone cleavage, and small-molecule inhibitor screening. It does not model membrane-anchored DPP-4 interactions with ADA or FAP on the cell surface.

What substrates does recombinant DPP-4 cleave and which fluorogenic substrate is best for activity assays?

DPP-4 (EC 3.4.14.5) cleaves N-terminal dipeptides from substrates bearing a penultimate proline, including GLP-1(7-36)NH2, GIP, SDF-1α/CXCL12, and NPY. For in vitro fluorogenic activity assays, H-Gly-Pro-AMC is the standard substrate: DPP-4 hydrolyzes the Gly-Pro bond to release free AMC (Ex 360 nm / Em 460 nm). A starting substrate concentration of 100–400 µM in assay buffer typically yields a linear fluorescence signal over 30–60 minutes at 37°C. This substrate is commercially available from several peptide suppliers and is selective enough for routine inhibitor IC50 determination with REC-DPP4.

What buffer conditions work best for DPP-4 activity assays and inhibitor IC50 screening?

REC-DPP4 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For fluorogenic cleavage assays, dilute into a working assay buffer of 25–50 mM Tris-HCl or HEPES, pH 7.4–8.0, 100–150 mM NaCl, 0.01–0.05% BSA or Tween-20 to reduce non-specific adsorption. The glycerol carry-over at typical enzyme dilutions is negligible. For IC50 determinations, pre-incubate enzyme with inhibitor for 15–30 minutes before substrate addition; DPP-4 inhibitors (e.g., sitagliptin, saxagliptin) show competitive kinetics, so substrate concentration relative to Km will shift apparent IC50 — factor this into experimental design.

What is the recommended starting enzyme concentration for a DPP-4 fluorogenic substrate cleavage assay?

A starting point of 2–10 nM REC-DPP4 in a 50–100 µL assay volume with 200 µM H-Gly-Pro-AMC typically produces a robust, linear fluorescence increase over 30–60 minutes at 37°C. Titrate enzyme concentration to confirm linearity — if signal saturates within the first 10 minutes, reduce enzyme 2–3 fold. For Km/Vmax determination, fix enzyme at the lowest concentration that gives a reliable signal-to-noise ratio (≥5:1 over background) and vary substrate from ~10 µM to 1 mM. Activity can be normalized to protein concentration using the supplied lot-specific activity data.

Can I use REC-DPP4 as a positive control for Western blot with the matched RP-DPP4 rabbit polyclonal antibody?

Yes — this is the primary intended pairing. RP-DPP4 (/anti-dpp-4-rabbit-polyclonal-antibody) is the matched rabbit polyclonal raised in the same lab against the DPP-4 extracellular domain, and compatibility with REC-DPP4 on Western blot is validated, not predicted. Load 20–50 ng of REC-DPP4 per lane alongside your cell lysate or plasma samples; expect a clean band at 90–110 kDa under reducing conditions. This controlled positive control eliminates ambiguity when troubleshooting faint or absent bands in endogenous DPP-4 Western blots, particularly in low-expressing tissues.

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

For Western blot with RP-DPP4, load 20–50 ng of REC-DPP4 per lane as a positive control. At 50 ng, the band at 90–110 kDa is reliably detectable with a standard HRP-conjugated secondary antibody at 1:5,000–1:10,000 dilution and ECL detection. If you are using a more sensitive detection system (e.g., fluorescent secondary, chemiluminescence with signal amplification), 10–20 ng per lane is sufficient and avoids signal oversaturation. Avoid loading >100 ng, which can cause band spreading and interfere with adjacent sample lanes on the same blot.

How should I store and handle REC-DPP4 to preserve enzymatic activity over time?

REC-DPP4 is supplied as a single-use aliquot in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, ready to use without reconstitution. Store at -20°C immediately upon receipt; the 10% glycerol cryoprotects enzymatic activity. Avoid repeated freeze-thaw cycles — each cycle can reduce activity by 10–20%. On the day of use, thaw on ice, briefly spin down, and dilute directly into assay buffer. For working dilutions, prepare fresh on the day of the experiment and keep on ice; do not leave diluted enzyme at room temperature for extended periods. Shelf life is 12 months from date of manufacture at -20°C.