Polyserase-1 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for Polyserase-1 recombinant on SDS-PAGE or Western blot?

The full-length Polyserase-1 polypeptide (1,059 aa; UniProt Q7Z410) has a predicted molecular weight of ~117 kDa, but HEK293-derived material runs at an apparently higher MW on SDS-PAGE due to N-linked glycosylation — expect a diffuse band in the 130–150 kDa range under denaturing conditions. Post-translational cleavage can also yield the individual Serase-1, Serase-2, and Serase-3 subunits depending on preparation conditions; confirm which form is present with the matched antibody RP-Polyserase1 and a reducing/non-reducing lane comparison.

Does recombinant Polyserase-1 require post-translational cleavage to be active, and which subunits are catalytically active?

Yes. Full-length Polyserase-1 undergoes post-translational proteolytic processing that releases three distinct serine protease subunits — Serase-1, Serase-2, and Serase-3 — each bearing a canonical His/Asp/Ser catalytic triad. Functional data show that Serase-1 and Serase-2 are catalytically active against fluorogenic substrates, while evidence for autonomous Serase-3 activity is limited. This recombinant is produced in HEK293 cells under conditions that support mammalian-type processing; lot-specific SDS-PAGE profiles document the cleavage state of each batch.

What fluorogenic substrates work best for a Polyserase-1 activity assay?

Serase-1 and Serase-2 hydrolyze N-t-Boc-Gln-Ala-Arg-AMC and N-t-Boc-Gln-Gly-Arg-AMC, both AMC-releasing substrates that reflect a trypsin-like, Arg/Lys-directed specificity. Monitor fluorescence at Ex/Em 380/460 nm. Substrates presenting Ala-Phe-Lys or Ala-Pro-Ala motifs are not appreciably cleaved, so avoid those as primary readouts. Start with substrate concentrations in the 100–200 µM range against 0.5–2 µg of recombinant protein per reaction and titrate to establish linear kinetics before running inhibitor panels.

What assay buffer should I use for Polyserase-1 serine protease activity assays?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with dilution directly into standard serine protease assay buffers (e.g., 50 mM Tris-HCl pH 8.0, 100–150 mM NaCl, 0.01% Tween-20). Keep glycerol carryover below 1% in the final reaction volume to avoid viscosity artifacts. Avoid EDTA or general metalloprotease inhibitor cocktails that contain serine protease inhibitors (e.g., AEBSF, PMSF); these will ablate activity. DTT at ≤1 mM is generally tolerated if reducing conditions are needed.

What starting concentration of recombinant Polyserase-1 should I use for an IC50 inhibitor assay?

For IC50 determinations, a starting enzyme concentration of 0.5–1 µg per 100 µL reaction is a practical entry point. Confirm you are in the linear range of substrate hydrolysis over the assay time window (typically 30–60 min at 37°C) before running compound titrations; enzyme titration experiments at 0.25, 0.5, 1, and 2 µg/reaction with 100 µM N-t-Boc-Gln-Ala-Arg-AMC will establish this. Adjust substrate concentration to ≤Km to ensure competitive inhibitor IC50 values approximate true Ki.

Can I use recombinant Polyserase-1 as a positive control for Western blot with the RP-Polyserase1 antibody?

Yes — this is the primary validated use case. RP-Polyserase1 (/anti-polyserase-1-rabbit-polyclonal-antibody) is raised in the same lab against Polyserase-1 and is guaranteed compatible with this recombinant for Western blot positive controls. Load 20–50 ng of REC-Polyserase1 per lane alongside your cell lysate samples. The antibody is validated for Western blot and detects the glycosylated band in the 130–150 kDa range. This pairing is also useful for antibody lot-to-lot validation and for confirming endogenous signal in tissues with predicted or validated Polyserase-1 expression.

How much recombinant Polyserase-1 should I load for a Western blot positive control lane?

20–50 ng per lane is sufficient for a clean signal with RP-Polyserase1 under standard ECL detection. If your lysate lanes require long exposure times, load on the lower end (20 ng) to prevent the recombinant control from saturating the film or CCD while the endogenous bands remain in the linear range. Run the positive control in a flanking lane rather than between lysate lanes to avoid any carry-over compression of the glycosylated, diffuse band at ~130–150 kDa.

How should I handle, aliquot, and store recombinant Polyserase-1 to preserve activity long-term?

REC-Polyserase1 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. Upon receipt, spin briefly, prepare single-use aliquots sized to your typical experiment (5–10 µg each), and store at -20°C. Avoid repeated freeze-thaw cycles — each cycle risks measurable loss of serine protease activity. Do not store at 4°C for more than 48 hours. If dilution below ~0.1 mg/mL is required for assays, add BSA (0.1%) to the working dilution to minimize adsorption to tube surfaces. Purity is >90% by SDS-PAGE; endotoxin is <0.1 EU/µg by LAL.