Elastase-2 (Recombinant)

Frequently Asked Questions

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

The mature human Elastase-2 (ELANE, UniProt P08246) has a predicted molecular weight of ~29 kDa based on its 267-amino-acid sequence. However, because this recombinant is expressed in HEK293 mammalian cells and carries native-like N-linked glycosylation, the observed band on SDS-PAGE typically migrates at approximately 33–36 kDa under reducing conditions. Under non-reducing conditions, disulfide-dependent compaction can shift apparent migration slightly. Always run a prestained marker in the 25–50 kDa range when confirming identity.

Is this recombinant Elastase-2 the mature processed form or does it include the propeptide?

This recombinant corresponds to the mature, active form of human Elastase-2 — the propeptide has been removed during processing, consistent with the physiologically relevant enzyme stored in neutrophil azurophil granules. It retains the canonical Ser-His-Asp catalytic triad (EC 3.4.21.37). Because propeptide-free material can autoactivate at neutral pH, we recommend keeping the enzyme on ice during setup and adding substrate immediately after dilution into assay buffer to avoid activity loss from autoproteolysis.

What substrates does recombinant Elastase-2 cleave, and which fluorogenic peptide is best for activity assays?

Elastase-2 efficiently hydrolyses the fluorogenic peptide MeOSuc-AAPV-AMC (ex/em ~360/460 nm), which is the most widely cited small-molecule substrate for kinetic characterization and inhibitor screening. Macromolecular substrates include elastin, fibronectin, and outer membrane protein A (OmpA). For routine activity confirmation or IC50 measurements, MeOSuc-AAPV-AMC at 100–200 µM in 50 mM Tris-HCl pH 7.5, 150 mM NaCl is a practical starting point. Substrate concentration should bracket the reported Km (~150 µM) to allow reliable Michaelis–Menten fitting.

What buffer conditions work best for Elastase-2 (Recombinant) activity assays, and can I use PBS?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is also a suitable assay buffer backbone. For activity assays, omit glycerol or dilute it to ≤1% to avoid viscosity effects on fluorescence readings. PBS (pH 7.4) is compatible but slightly suboptimal; Elastase-2 activity is pH-sensitive, and peak activity is typically reported between pH 7.4–8.0. Avoid buffers containing primary amines (e.g., Tris above 100 mM at low enzyme concentrations) or reducing agents such as DTT or TCEP, which can disrupt disulfide bonds critical for catalytic geometry.

What starting enzyme concentration should I use for a recombinant Elastase-2 activity or inhibition assay?

A starting concentration of 1–10 nM recombinant Elastase-2 in assay buffer is appropriate for fluorogenic peptide cleavage assays using MeOSuc-AAPV-AMC at 100–200 µM. For macromolecular substrate assays (elastin, fibronectin), 50–200 nM is more typical to observe meaningful turnover within a 30–60 min window. For IC50 determinations, keep enzyme concentration at or below the Ki of your inhibitor to avoid tight-binding artifacts — generally 2–5 nM works well for known neutrophil elastase inhibitors. Titrate in pilot experiments before committing to a full inhibitor panel.

Can I use recombinant Elastase-2 as a Western blot positive control with the matched anti-Elastase-2 antibody?

Yes — this is one of the primary intended uses of REC-Elastase2 alongside the matched rabbit polyclonal antibody (SKU: RP-Elastase2, available at /anti-elastase-2-rabbit-polyclonal-antibody). Because both are produced and validated in the same laboratory, epitope compatibility is confirmed rather than assumed. Load 20–50 ng of recombinant protein per lane under reducing conditions; you should detect a clear band at ~33–36 kDa with RP-Elastase2. This pairing is especially useful when setting up detection in new cell or tissue lysates where endogenous expression levels are uncertain.

How much recombinant Elastase-2 should I load for a Western blot positive control, and what dilution of RP-Elastase2 antibody should I use?

Load 20–50 ng of REC-Elastase2 per lane for a reliable positive control band at ~33–36 kDa. For the matched rabbit polyclonal RP-Elastase2, a primary antibody dilution of 1:1,000–1:2,000 in 5% non-fat dry milk or BSA/TBST typically yields a clean signal with minimal background in our hands. Exposure times will vary by detection system, but with HRP-conjugated secondary antibodies and chemiluminescent substrate, a 30–60 second exposure is usually sufficient at the 20–50 ng loading range. Reduce loading to 5–10 ng if signal is saturating.

How should I store and handle recombinant Elastase-2 to maintain activity, and what is the shelf life?

REC-Elastase2 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. Avoid repeated freeze-thaw cycles — each cycle can cause measurable loss of serine protease activity due to partial unfolding at the active site. Once thawed, keep on ice and use within 4–8 hours. When stored correctly, activity is stable for at least 12 months from the date of receipt. For dilute working stocks (<10 nM), add carrier protein (0.1% BSA) to the dilution buffer to prevent adsorptive losses to tube walls.