Marapsin-1 (Recombinant)

Frequently Asked Questions

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

The human PRSS27 open reading frame encodes 290 amino acids, giving a predicted unglycosylated MW of approximately 32 kDa. Because this recombinant is produced in HEK293 cells, N-linked glycosylation adds mass, so the predominant band on reducing SDS-PAGE typically runs at 35–42 kDa. Under non-reducing conditions, disulfide-dependent compaction can shift apparent mobility slightly. If you are running a Western blot positive control alongside RP-Marapsin1, expect the band in the 35–42 kDa window; load 50–100 ng per lane for a clean, unambiguous signal.

Is recombinant Marapsin-1 supplied as a zymogen or as the mature active form of PRSS27?

PRSS27 is natively secreted as a zymogen and undergoes proteolytic cleavage of its propeptide to yield the catalytically active serine protease. The REC-Marapsin1 recombinant is produced in HEK293 cells under conditions that favor secretion-coupled processing, and the material is supplied as the active enzyme form. That said, propeptide cleavage efficiency can vary batch to batch — the Certificate of Analysis specifies the processing status and includes an activity confirmation using a fluorogenic substrate. If zymogen-to-enzyme conversion is critical to your assay design, contact us for the lot-specific data before ordering.

What substrates does Marapsin-1 cleave, and what fluorogenic substrate should I use for an activity assay?

Marapsin-1 is a chymotrypsin-like serine protease (EC 3.4.21.-), meaning it preferentially cleaves after bulky hydrophobic residues — phenylalanine, tyrosine, and leucine. For fluorogenic activity assays, Suc-Ala-Ala-Pro-Phe-AMC is a well-validated chymotrypsin-like substrate and provides a strong signal with this enzyme class. Alternatively, Z-Phe-Arg-AMC can be tested if you wish to probe broader specificity. Run reactions at 37°C in 50 mM Tris-HCl pH 7.5, 150 mM NaCl — matching the storage buffer — and monitor fluorescence at Ex 380 nm / Em 460 nm. Include a serine protease inhibitor control (e.g., PMSF or AEBSF) to confirm signal specificity.

What buffer conditions and starting enzyme concentration should I use for a Marapsin-1 protease activity assay?

Start in 50 mM Tris-HCl pH 7.5, 150 mM NaCl — this matches the storage buffer, so no buffer-exchange step is required for initial characterization. A starting enzyme concentration of 1–5 nM is reasonable for fluorogenic substrate assays using Suc-Ala-Ala-Pro-Phe-AMC at 100 µM substrate. Optimize pH between 7.0–8.5 if you need to mimic a specific tissue microenvironment; chymotrypsin-like proteases are generally most active at slightly alkaline pH. Avoid Ca²⁺ chelators such as EDTA unless you are intentionally testing ionic effects. Include 0.01% BSA to minimize non-specific adsorption to low-binding plates.

How do I set up an inhibitor IC50 assay with recombinant Marapsin-1, and what controls should I include?

For IC50 determination, use 1–2 nM REC-Marapsin1 and 100 µM Suc-Ala-Ala-Pro-Phe-AMC in 50 mM Tris-HCl pH 7.5, 150 mM NaCl. Pre-incubate enzyme with inhibitor for 30 minutes at room temperature before adding substrate to allow equilibration, particularly for slow-binding inhibitors. Measure initial velocity (first 5–10 minutes of linear phase) rather than endpoint fluorescence. Include PMSF or AEBSF as a positive inhibition control (complete inhibition expected at 1 mM), substrate-only blank, and enzyme-only vehicle control. Fit velocity-vs.-concentration data to a four-parameter logistic model to extract IC50.

Can I use REC-Marapsin1 as a Western blot positive control for the RP-Marapsin1 rabbit polyclonal antibody?

Yes — this is one of the primary intended uses of REC-Marapsin1. The RP-Marapsin1 rabbit polyclonal antibody (catalog /anti-marapsin-1-rabbit-polyclonal-antibody) was raised against the same PRSS27 sequence used for this recombinant, so epitope compatibility is guaranteed. Load 50–100 ng of REC-Marapsin1 per lane alongside your experimental lysates; expect a band at 35–42 kDa given HEK293-derived glycosylation. This pairing is also useful for antibody lot-to-lot validation: consistent band intensity and mobility across lots confirms antibody performance without relying on variable endogenous expression in cell lines.

How much recombinant Marapsin-1 should I load per lane for a Western blot positive control, and what dilution of RP-Marapsin1 antibody should I use?

Load 50–100 ng of REC-Marapsin1 per lane for a strong, clean positive control band at 35–42 kDa. For the matched RP-Marapsin1 rabbit polyclonal antibody, a primary antibody dilution of 1:500–1:2,000 in 5% non-fat milk or 3% BSA/TBST is a practical starting range for Western blot; optimize for your blocking reagent and membrane type. Because the recombinant is >90% pure by SDS-PAGE and the antibody was generated against the same PRSS27 construct, background from off-target bands is minimal at these dilutions. Reduce antibody concentration if background is high, rather than reducing protein load.

How should I store recombinant Marapsin-1, and what is its expected shelf life after thawing?

REC-Marapsin1 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and shipped on dry ice. Store at -20°C in the single-use aliquots provided. Avoid repeated freeze-thaw cycles — each cycle can reduce serine protease activity by 10–20% through denaturation and aggregation. Once thawed, keep on ice and use within 4–8 hours; do not refreeze a working aliquot. Under correct storage, activity and integrity are stable for 12 months from the date of receipt. If you anticipate using the protein across multiple sessions, prepare smaller working aliquots immediately after the first thaw.