Proteinase-3 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant Proteinase-3 on SDS-PAGE or Western blot?

The mature, processed form of PR-3 (PRTN3; UniProt P24158) runs at approximately 29–32 kDa under reducing SDS-PAGE conditions. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation is present, which typically shifts the apparent MW 2–4 kDa above the theoretical 29 kDa of the 256-aa polypeptide. Expect a diffuse band in the 29–34 kDa range. Under non-reducing conditions, the disulfide-bonded form migrates slightly differently, so always run a reducing control when confirming identity by Western blot.

What processing form of Proteinase-3 is this recombinant — pro-form or mature active enzyme?

This product (REC-Proteinase3) is the mature, active form of PR-3, not the zymogen pro-form. The signal peptide and propeptide sequences have been removed during HEK293 expression and processing, yielding a secreted, catalytically competent serine protease (EC 3.4.21.76). This matters for activity assays: you do not need an exogenous activation step before use. If your experiment requires the zymogen as a negative control, this product is not the appropriate reagent — contact us to discuss options.

What substrates can recombinant Proteinase-3 cleave and which are best for in vitro activity assays?

PR-3 has a broad extracellular matrix substrate repertoire including elastin, fibronectin, laminin, vitronectin, and collagen types I, III, and IV — useful for multi-substrate MS-based cleavage profiling. For quantitative activity assays, synthetic chromogenic or fluorogenic peptide substrates are preferred. MeOSuc-AAPV-pNA (chromogenic) and MeOSuc-AAPV-AMC (fluorogenic) are well-validated PR-3 substrates; fluorogenic formats typically offer better sensitivity at lower enzyme concentrations. Confirm substrate Km under your specific buffer conditions, as ionic strength influences Km for small peptide substrates.

What buffer conditions work best for Proteinase-3 activity assays and does the storage buffer interfere?

PR-3 is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For activity assays, the storage buffer is generally compatible as a working buffer at ≥10-fold dilution, bringing glycerol below 1% v/v — concentrations above ~5% glycerol can suppress catalytic rates. Most published PR-3 assays use 50–100 mM HEPES or Tris pH 7.4–8.0 with 150 mM NaCl and 0.01–0.05% Tween-20 to reduce surface adsorption. Avoid EDTA; PR-3 is a serine protease, not a metalloprotease, but calcium at low concentrations can stabilize the active site.

What starting concentration of recombinant Proteinase-3 should I use for an IC50 inhibitor assay?

For IC50 determinations against serine protease inhibitors, a common starting point is 1–10 nM PR-3 in assay buffer, used well below the Km of your fluorogenic substrate to satisfy the assumptions of the Cheng-Prusoff equation. Pilot experiments should confirm linearity of substrate hydrolysis over the assay time window at your chosen enzyme concentration. REC-Proteinase3 is supplied at >90% purity by SDS-PAGE and <0.1 EU/µg endotoxin, so background hydrolysis from contaminants is minimal — important when measuring inhibition at low nM enzyme concentrations.

Can I use REC-Proteinase3 as a positive control for Western blot with the matched RP-Proteinase3 antibody?

Yes — this is the recommended use case. REC-Proteinase3 is produced in the same laboratory as the matched rabbit polyclonal antibody (SKU: RP-Proteinase3; see /anti-proteinase-3-rabbit-polyclonal-antibody), and compatibility for Western blot has been validated with this antigen-antibody pair. Load 25–100 ng of REC-Proteinase3 per lane for a clear positive control band at 29–34 kDa. Because both reagents originate from the same source material, this pairing eliminates one of the most common sources of antibody validation ambiguity — antigen mismatch between immunogen and test sample.

How much recombinant Proteinase-3 should I load for Western blot as a positive control lane?

Load 25–100 ng per lane as a starting range with the matched RP-Proteinase3 antibody (SKU: RP-Proteinase3). At 50 ng, the 29–34 kDa band is reliably detected under standard ECL conditions with the antibody diluted 1:1,000–1:5,000. If you are running lysates from PR-3-expressing cells (e.g., neutrophil lysates) alongside, a 50 ng recombinant lane provides a clean, unambiguous size reference. Reduce to 25 ng if your secondary antibody or detection system is particularly sensitive to avoid saturation of the positive control band.

How should I store and handle REC-Proteinase3 to preserve enzymatic activity over time?

Store at −20°C in the supplied single-use aliquots (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol). Avoid repeated freeze-thaw cycles — each cycle can reduce specific activity by measurable increments for serine proteases with multiple disulfide bonds. On the day of use, thaw on ice and dilute directly into pre-warmed assay buffer. For dilutions below ~10 µg/mL, add 0.1% BSA (protease-free) as a carrier to prevent adsorption losses to tube walls. Shelf life is ≥12 months from receipt under recommended storage conditions.