Marapsin-2 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Marapsin-2 on SDS-PAGE and Western blot?

Marapsin-2 (PRSS38; UniProt A1L453) has a predicted unmodified molecular weight of approximately 36 kDa based on its 326-amino-acid sequence. Because this recombinant is produced in HEK293 mammalian cells, N-linked glycosylation is expected to shift the apparent MW on SDS-PAGE to approximately 40–45 kDa under reducing conditions. The pro-domain, if partially retained, may produce a doublet; the mature catalytic form runs at the lower end of that range. Always run alongside a broad-range molecular weight marker and confirm identity with RP-Marapsin2 if band identity is ambiguous.

Is recombinant Marapsin-2 supplied as the zymogen or the mature active form, and how is it processed?

The recombinant is supplied as the active, mature enzyme form. HEK293 expression supports endogenous pro-domain processing pathways, so the secreted product has undergone cleavage of the inhibitory pro-peptide and represents the catalytically competent serine protease. This is important because incorrectly processed zymogen forms can be enzymatically silent. The canonical Ser-His-Asp catalytic triad is intact and oriented for substrate engagement. If your experimental design specifically requires the zymogen, note that this product is not the appropriate format — contact us for guidance on generating inhibited forms chemically post-purchase.

What substrates are recommended for a Marapsin-2 activity assay and what fluorogenic peptide should I start with?

Marapsin-2 is a chymotrypsin-like serine endopeptidase (EC 3.4.21.-) with predicted preference for bulky hydrophobic residues at the P1 position (Phe, Tyr, Trp, Leu). A reasonable starting fluorogenic substrate is Suc-Ala-Ala-Pro-Phe-AMC (100 µM), which is well-characterized for chymotrypsin-family proteases. Measure fluorescence at Ex/Em 360/460 nm. Because published kinetic constants for Marapsin-2 specifically are limited, run a substrate concentration series (25–400 µM) to establish Km empirically before reporting kcat/Km values. Include PMSF (1 mM) or AEBSF as a serine protease inhibitor control to confirm signal is enzyme-dependent.

What buffer conditions are optimal for Marapsin-2 protease activity assays?

The protein is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and is compatible with direct use in activity assays at physiologically relevant pH (7.2–7.6). For activity assays, dilute into assay buffer containing 50 mM Tris-HCl pH 7.5, 100–150 mM NaCl, 0.01–0.05% Tween-20 or BSA (0.1%) to minimize adsorption to assay plate surfaces. Avoid EDTA — it is not required for this serine protease and can interfere with some co-factor-dependent readouts. Glycerol from the storage buffer is diluted to ≤1% in the assay, which is generally well-tolerated. Keep assay temperature consistent at 37°C for reproducibility.

What starting enzyme concentration should I use for a recombinant Marapsin-2 fluorogenic substrate cleavage assay?

In the absence of established published kinetic data specific to Marapsin-2, start with 50–200 nM recombinant enzyme in a 100 µL assay volume alongside a Suc-Ala-Ala-Pro-Phe-AMC substrate titration. Measure initial velocity (first 10–20% substrate conversion) to remain in the linear range. If signal is low at 50 nM, step up to 200–500 nM; if substrate is depleted rapidly, reduce enzyme concentration accordingly. For IC50 experiments with serine protease inhibitors, hold enzyme concentration at or below Km to avoid substrate-competition artifacts. Titrate enzyme linearity before committing to inhibitor screening runs.

Can I use recombinant Marapsin-2 as a positive control for Western blot with the RP-Marapsin2 rabbit polyclonal antibody?

Yes — this is a validated use case. Recombinant Marapsin-2 (REC-Marapsin2) and the matched rabbit polyclonal antibody (RP-Marapsin2) are produced and validated in the same laboratory, ensuring antigen-antibody compatibility. Load 50–100 ng of recombinant protein per lane under reducing SDS-PAGE conditions; expect a band at approximately 40–45 kDa due to glycosylation. This serves as both a size reference and a signal calibration point alongside tissue or cell lysate lanes. RP-Marapsin2 is validated for Western blot detection, so the recombinant positive control directly confirms antibody performance and correct band identification in your experimental system.

How much recombinant Marapsin-2 should I load as a Western blot positive control to get a clean band without overloading?

Load 50–100 ng per lane as a starting point when using RP-Marapsin2 at its recommended dilution range. At 50 ng, the band is typically clean and distinct without smearing; 100 ng provides a stronger signal useful for optimizing exposure or confirming antibody sensitivity at lower primary antibody concentrations. Avoid loading more than 200–250 ng, as overloading can cause trailing and obscure doublet resolution between pro- and mature forms. Run the positive control lane adjacent to your most critical experimental lane so exposure matching is straightforward. Use Coomassie staining on a parallel gel to confirm loading consistency.

How should I store and handle recombinant Marapsin-2 to maintain activity over time, and what is its shelf life?

Recombinant Marapsin-2 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. Avoid repeated freeze-thaw cycles, as these progressively denature the active site and reduce specific activity. Shelf life is ≥12 months at −20°C in unopened aliquots. Once thawed, keep on ice and use within 4–8 hours; do not re-freeze. If working dilutions are needed across multiple experiments, prepare small secondary aliquots in assay-compatible buffer with 0.1% BSA as carrier, store at −80°C, and use within 1 month. Endotoxin is certified <0.1 EU/µg by LAL assay.