PRSS-56 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant PRSS-56 on SDS-PAGE and Western blot?

The PRSS-56 open reading frame encodes 603 amino acids, giving a predicted unmodified MW of ~67 kDa. Because this recombinant is produced in HEK293 cells and undergoes mammalian N-linked glycosylation, the apparent MW on SDS-PAGE typically runs at 75–85 kDa — a shift consistent with glycoprotein behaviour. On Western blot under reducing conditions, expect a diffuse band in that 75–85 kDa window. Purity is confirmed >95% by SDS-PAGE prior to release. If you are using our matched antibody RP-PRSS56 for the blot, this band serves as your positive control reference.

Is recombinant PRSS-56 supplied as a zymogen or as the mature processed form?

PRSS-56 is a serine protease that undergoes zymogen maturation; the HEK293 expression system used here supports signal-peptide cleavage and the post-translational folding required for correct disulfide architecture. The recombinant supplied by Triple Point Biologics is the processed, active-enzyme form — not the uncleaved zymogen. This is relevant if you are running substrate-cleavage or inhibitor assays where pre-activation steps would otherwise introduce variability. The active form also means you can proceed directly to kinetic characterisation without an in vitro activation protocol.

What fluorogenic substrate should I use to measure PRSS-56 serine protease activity?

PRSS-56 is classified under EC 3.4.21.- (serine endopeptidases). For initial activity screening, synthetic fluorogenic substrates with a Arg-AMC or Lys-AMC scaffold (e.g., Boc-Phe-Ser-Arg-AMC or H-Arg-AMC) are a reasonable starting point given the trypsin-like cleavage preference common to this class. Monitor release of AMC fluorescence at Ex 380 nm / Em 460 nm. Because PRSS-56's precise substrate preference is still being characterised in the literature, we recommend running a substrate panel at 100–200 µM substrate concentration against 50–200 nM enzyme to identify highest-signal hits before committing to kinetic measurements.

What assay buffer conditions work best for PRSS-56 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 at low dilution, provided glycerol carry-over does not exceed ~1% in the final reaction volume. For kinetic assays (Km, kcat determination), dilute enzyme into a working buffer of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20 to minimise surface adsorption at low enzyme concentrations. Avoid Ca²⁺ chelators (EGTA, EDTA) unless your experimental design requires them. Maintain pH between 7.0 and 8.0; serine protease activity typically drops sharply below pH 6.5.

What starting enzyme concentration should I use for an IC50 inhibitor screen against recombinant PRSS-56?

For a well-behaved IC50 determination, enzyme concentration in the assay well should be at or below the anticipated Ki of your inhibitor scaffold — typically 10–50 nM recombinant PRSS-56 is a practical starting range. Run an activity-versus-concentration curve first (1–500 nM) to identify a linear signal window. Substrate should be held below Km (empirically determined) to satisfy the Morrison tight-binding assumptions if you expect sub-nanomolar inhibitors. Pre-incubate enzyme with inhibitor for 30 min at room temperature before substrate addition to allow equilibrium binding. Stock concentration of REC-PRSS56 should be verified by A280 or BCA before the screen.

Can I use recombinant PRSS-56 (REC-PRSS56) as a positive control for Western blot with the matched antibody RP-PRSS56?

Yes — this is one of the primary use cases for REC-PRSS56 alongside RP-PRSS56. The rabbit polyclonal antibody RP-PRSS56 was validated against this recombinant in-house, so the two reagents are guaranteed compatible for Western blot positive-control applications. Load 50–100 ng of REC-PRSS56 per lane under reducing conditions; you should see a clean band at 75–85 kDa with RP-PRSS56 at its recommended primary antibody dilution. This also makes REC-PRSS56 a reliable standard for antibody lot-to-lot consistency checks and for validating antibody performance in new cell or tissue lysate backgrounds.

How much recombinant PRSS-56 should I load for a Western blot positive control lane?

50–100 ng per lane is sufficient for a strong, clean signal with RP-PRSS56 on standard PVDF or nitrocellulose membranes. If you are optimising antibody dilution simultaneously, running a titration of 10, 25, 50, and 100 ng across adjacent lanes gives a useful signal-linearity check. Resolve on a 10% or gradient (8–12%) SDS-PAGE gel to best resolve the 75–85 kDa glycosylated band. At >200 ng the band can appear diffuse due to glycan heterogeneity, which is normal for HEK293-expressed glycoproteins and does not indicate protein degradation.

How should I store and handle recombinant PRSS-56 to preserve enzymatic activity long-term?

REC-PRSS56 is shipped in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and arrives ready to use — no reconstitution required. Store at -20°C in the single-use aliquots provided; repeated freeze-thaw cycles progressively reduce activity and should be avoided. For working stocks used within a week, storage at 4°C is acceptable, but activity loss beyond 5–7 days at 4°C has been observed for serine proteases of this class. Add BSA (0.05–0.1 mg/mL) to diluted working solutions to reduce adsorptive losses at low enzyme concentrations (<10 nM). Do not heat above 37°C during assay setup.