Polyserase-2 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant Polyserase-2 on SDS-PAGE or Western blot?

The full-length human Polyserase-2 (PRSS36, UniProt Q5K4E3) is 855 amino acids, giving a predicted polypeptide molecular weight of approximately 95 kDa. Because this recombinant is produced in HEK293 cells, N-linked glycosylation adds additional mass; the apparent MW on reducing SDS-PAGE typically runs higher, often in the 110–130 kDa range depending on gel conditions and glycosylation extent. If you are comparing to a bacterially expressed standard, expect a meaningful upward shift. Under non-reducing conditions, disulfide-mediated dimers may also be visible.

Which isoform or processing form of Polyserase-2 does this recombinant represent — full-length or a domain fragment?

This product represents the secreted, full-length human Polyserase-2 (855 aa, PRSS36). The native protein is processed post-translationally: the signal peptide is removed upon secretion, and the catalytic serine protease domain is retained in the mature form. Production in HEK293 cells allows proper signal-peptide cleavage and disulfide-bond formation, so the recombinant closely mirrors the extracellular, matrix-associated species you would detect in conditioned medium or ECM fractions. If your experimental model involves intracellular forms, this product is not the appropriate reference.

What fluorogenic substrates work best for a Polyserase-2 activity assay?

Polyserase-2 preferentially cleaves substrates carrying Arg at the P1 position. The two highest-efficiency fluorogenic substrates are N-t-Boc-Gln-Ala-Arg-AMC and N-t-Boc-Gln-Gly-Arg-AMC. Both yield significantly greater fluorescent signal per unit time than the corresponding Lys-P1 substrates (N-t-Boc-Ala-Phe-Lys-AMC and N-t-Boc-Val-Leu-Lys-AMC). For initial activity verification, N-t-Boc-Gln-Ala-Arg-AMC at 100–200 µM in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) is a practical starting point. Monitor AMC release at Ex/Em 360/460 nm.

What buffer conditions should I use for a Polyserase-2 fluorometric kinetics assay?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with direct use in activity assays after a simple dilution step to reduce glycerol below 1% (v/v) in the final reaction volume. Glycerol above ~2% can suppress fluorogenic substrate turnover. Maintain pH 7.5 and include 0.01% BSA or 0.01% Tween-20 to minimize surface adsorption at low enzyme concentrations. Polyserase-2 is a serine protease; include a PMSF or AEBSF negative-control condition to confirm on-target activity and distinguish from any contaminating protease activity.

What starting concentration of recombinant Polyserase-2 should I use in an inhibitor IC50 assay?

For a fluorometric IC50 assay, begin with 1–5 nM recombinant Polyserase-2 in a 100 µL reaction volume, using N-t-Boc-Gln-Ala-Arg-AMC at a substrate concentration bracketing your estimated Km (typically 50–200 µM for Arg-AMC substrates). Confirm that enzyme concentration is within the linear range of signal generation before running compound dose-responses. The protein is supplied at >90% purity by SDS-PAGE and <0.1 EU/µg endotoxin, so LPS interference in cell-based counter-screens is not a primary concern at these concentrations.

How much recombinant Polyserase-2 should I load as a positive control for Western blot?

Load 50–200 ng per lane as a positive control alongside your lysate samples. At 100 ng, the band at ~110–130 kDa (apparent MW, glycosylated) is reliably detected with RP-Polyserase2 (our matched rabbit polyclonal antibody, /anti-polyserase-2-rabbit-polyclonal-antibody) at a 1:500–1:2000 dilution. Because the recombinant runs above the predicted 95 kDa due to glycosylation, annotate this clearly in figure legends to avoid reviewer confusion. Use reducing conditions to collapse any disulfide-linked species to a single predominant band.

Can I use recombinant Polyserase-2 as a positive control when validating the RP-Polyserase2 rabbit polyclonal antibody?

Yes, and this is the intended paired use. REC-Polyserase2 and RP-Polyserase2 are produced in the same lab and validated together for Western blot. Load 50–200 ng of the recombinant alongside cell or tissue lysates known to express PRSS36. The recombinant provides an unambiguous positive-control band to confirm antibody specificity and appropriate molecular weight, which is especially useful when endogenous expression in your cell line is low or uncertain. RP-Polyserase2 is also validated for IHC, where the recombinant can serve as a spike-in control in solution-phase blocking experiments.

How should I store and handle recombinant Polyserase-2 to preserve enzymatic activity long-term?

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) stabilizes activity during frozen storage; the glycerol prevents freeze-concentration damage. Avoid repeated freeze-thaw cycles — each cycle can cause measurable loss of specific activity in serine proteases. For working dilutions, prepare fresh in assay buffer on ice and use within 4–8 hours. Do not dilute to below ~0.1 µg/mL without a carrier protein (0.1% BSA), as adsorptive losses to tube walls become significant at very low concentrations.