DPP-8 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant DPP-8 on SDS-PAGE and Western blot?

Full-length human DPP-8 (898 aa, UniProt Q6V1X1) has a predicted molecular weight of ~100 kDa from sequence alone. Because this recombinant is produced in HEK293 cells, N-linked glycosylation is expected to shift the apparent band to approximately 100–115 kDa on reducing SDS-PAGE. Purity is >90% by SDS-PAGE, so the dominant band at that position should be unambiguous. When running a Western blot positive control alongside RP-DPP8 antibody, load 20–50 ng per lane and expect a clean band in that 100–115 kDa window.

Does recombinant DPP-8 require any autocatalytic processing or is it active as a full-length protein?

Unlike some cysteine proteases that require zymogen activation, DPP-8 is active as the full-length 898-amino acid monomer — no propeptide cleavage is necessary. The catalytic triad (Ser, Asp, His) is constitutively accessible in the folded prolyl oligopeptidase (S9B) architecture. HEK293 expression ensures the cytoplasmic folding context required for this architecture. The recombinant is supplied in active solution form and should be functional immediately upon thaw, without any pre-activation step.

What substrates work best for measuring DPP-8 dipeptidyl peptidase activity in a fluorescence assay?

The two most widely validated fluorogenic substrates for DPP-8 are Gly-Pro-AMC and Ala-Pro-AMC, both cleaved at the Pro-AMC bond to release 7-amino-4-methylcoumarin (AMC), monitored at Ex/Em 360/460 nm. Gly-Pro-AMC is the conventional choice and allows direct comparison with published Km values for DPP4 and DPP9. Use substrate concentrations spanning 50–500 µM to bracket the Km for Km/kcat determination. H-Ala-Pro-pNA (chromogenic) is an alternative if fluorescence interference is a concern in your compound library.

What buffer conditions should I use to measure DPP-8 activity in a kinetic assay?

DPP-8 is active across a broad pH range, with optimal activity typically reported between pH 7.0 and 8.0. A practical starting buffer is 50 mM HEPES pH 7.5, 150 mM NaCl, 0.01% Tween-20, 1 mg/mL BSA at 37 °C. The 10% glycerol in the supplied storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is diluted to ≤1% final in the assay to avoid viscosity effects. Avoid EDTA — DPP-8 activity is not metal-dependent, but chelators can interfere with assay stability at high concentrations.

How much recombinant DPP-8 should I use per well for an IC50 inhibitor profiling assay?

For a 96-well fluorescence kinetic assay with Gly-Pro-AMC (200 µM), a starting point of 5–20 nM recombinant DPP-8 per well (in 100 µL total volume) typically gives a robust signal-to-background ratio of ≥5 with a linear rate for 30–60 minutes at 37 °C. Titrate enzyme concentration in a preliminary linearity-of-signal experiment before committing compound. Pre-incubate enzyme with inhibitor for 30 minutes at room temperature before substrate addition to allow equilibrium binding, particularly important for slow-binding inhibitors.

Can I use DPP-8 (Recombinant) as a positive control for Western blot with the RP-DPP8 antibody?

Yes — this is a validated pairing. REC-DPP8 and RP-DPP8 (rabbit polyclonal, /anti-dpp-8-rabbit-polyclonal-antibody) are developed in the same laboratory, and compatibility for Western blot positive control use is confirmed. Load 20–50 ng of REC-DPP8 in a dedicated lane alongside your cell lysate. Under reducing conditions, the primary band should appear at approximately 100–115 kDa. This is particularly useful when working with cell lines where endogenous DPP-8 expression is low or variable, giving you a definitive size reference and antibody sensitivity benchmark.

How much recombinant DPP-8 protein should I load for a Western blot positive control lane?

20–50 ng per lane is a reliable starting range when using RP-DPP8 at a 1:500–1:2000 primary antibody dilution on standard PVDF or nitrocellulose membranes. If your lysate lanes are loaded at 20–30 µg total protein, 20 ng of REC-DPP8 is generally sufficient to produce a visible, non-saturating band without overwhelming adjacent lanes. Titrate down to 5 ng if you are optimizing signal linearity for quantitative Western applications. The >90% purity by SDS-PAGE ensures the signal reflects DPP-8 specifically, not a mixed-protein background.

How should I store and handle DPP-8 (Recombinant) to preserve enzymatic activity over time?

REC-DPP8 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 immediately upon receipt. Repeated freeze-thaw cycles progressively reduce specific activity — even two additional cycles can measurably impair kinetic performance for an enzyme of this complexity. For experiments running over several days, keep a working aliquot at 4 °C for up to 48 hours. Avoid diluting to very low concentrations (<1 nM) without carrier protein (0.1–1 mg/mL BSA), as surface adsorption losses become significant at those ranges.