Elastase-3-A (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant CELA3A on SDS-PAGE and Western blot?

The full 270-amino acid sequence of CELA3A (UniProt P09093) predicts an unglycosylated molecular weight of approximately 29 kDa. Because this recombinant is produced in HEK293 cells, native-like glycosylation is retained, and the observed band on reducing SDS-PAGE typically migrates at 32–36 kDa. Under non-reducing conditions, intramolecular disulfide bonds may cause a slight upward shift. If you are running a Western blot positive control alongside RP-Elastase3A antibody, load the 32–36 kDa window as your reference.

What is the substrate specificity of CELA3A and how does it differ from neutrophil elastase?

CELA3A is a serine protease with a strong preference for alanine at the P1 position of its substrates, which makes it mechanistically distinct from neutrophil elastase (ELANE), whose primary P1 preference is valine or alanine on insoluble elastin matrices. CELA3A also differs from its closest paralog, CELA3B, in substrate selectivity. This P1-Ala preference means synthetic substrates such as Suc-Ala-Ala-Ala-pNA or Suc-Ala-Pro-Ala-pNA are appropriate for activity assays, while substrates optimized for ELANE (e.g., MeOSuc-AAPV-pNA) will underestimate CELA3A activity.

What activity assay substrate and buffer conditions work best for recombinant Elastase-3-A?

For chromogenic assays, Suc-Ala-Ala-Ala-pNA (3–5 mM final concentration) in 50 mM Tris-HCl pH 8.0, 150 mM NaCl provides a reliable baseline for CELA3A activity. The supplied storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible with direct dilution into assay buffer; the glycerol concentration will be negligible at typical working dilutions of 1:10 or greater. Monitor absorbance at 405 nm. A starting enzyme concentration of 50–200 nM is recommended for kinetic runs, titrating downward if substrate turnover is too rapid within the linear range.

What starting concentration of recombinant CELA3A should I use for an inhibitor IC50 experiment?

For IC50 determinations, use CELA3A at a concentration at or below the apparent Km for your chosen substrate to ensure inhibitor potency is not underestimated (Cheng-Prusoff correction). With Suc-Ala-Ala-Ala-pNA, a starting enzyme concentration of 50–100 nM is practical with substrate at 0.5–1 mM. Include a vehicle control and confirm linear product accumulation over the assay window (typically 10–30 min at 37°C). Because this enzyme is supplied at >90% purity by SDS-PAGE, active-site titration with a serine protease inhibitor (e.g., PMSF titration) is recommended to determine the fraction of catalytically competent enzyme before IC50 calculations.

Can recombinant Elastase-3-A be used as a positive control for Western blot with the matched RP-Elastase3A antibody?

Yes — this is a primary validated use case. REC-Elastase3A and RP-Elastase3A are developed from the same target sequence in the same laboratory, making them a guaranteed matched pair for Western blot positive controls and antibody validation experiments. Load 20–50 ng of REC-Elastase3A per lane under reducing SDS-PAGE conditions; the RP-Elastase3A polyclonal antibody is validated for Western blot and should detect a clean band at 32–36 kDa. This pairing is particularly useful when validating RP-Elastase3A against pancreatic tissue lysates or cell lines with exocrine pancreatic lineage.

How much recombinant Elastase-3-A should I load as a Western blot positive control, and what band should I expect?

Load 20–50 ng of REC-Elastase3A per lane on a 12% SDS-PAGE gel under reducing conditions. Expect a primary band at 32–36 kDa, reflecting the glycosylated, processed form produced in HEK293 cells. A faint higher-molecular-weight smear is occasionally observed and is consistent with heterogeneous glycosylation, not degradation — purity is >90% by SDS-PAGE. If background is elevated with RP-Elastase3A antibody, titrate the antibody concentration down rather than increasing the recombinant load; excess antigen can paradoxically increase non-specific signal in polyclonal blotting systems.

How should recombinant Elastase-3-A be stored and handled to preserve activity after delivery?

REC-Elastase3A is shipped in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol in single-use aliquots. Upon receipt, store immediately at -20°C. Do not refreeze after thawing; plan your aliquot sizes around single experimental sessions. For dilutions below 10 nM (e.g., binding or low-end activity assays), supplement the working buffer with 0.1% BSA to prevent adsorptive loss on tube surfaces. The enzyme retains activity for at least 12 months at -20°C when freeze-thaw cycles are avoided. Avoid detergents above 0.01% Tween-20, which can disrupt the active site of serine proteases at low enzyme concentrations.

Is recombinant CELA3A expressed as a zymogen or active enzyme, and does it require activation before use?

CELA3A is secreted by the exocrine pancreas as a zymogen (pro-CELA3A) that requires tryptic cleavage for activation in vivo. The REC-Elastase3A recombinant is provided in its active, processed form — the propeptide has been removed during manufacturing, so no additional activation step is required before activity assays. The HEK293 expression system supports correct disulfide bond formation, which is essential for the structural integrity of the chymotrypsin fold and, by extension, catalytic competence. Confirm activity upon receipt using the Suc-Ala-Ala-Ala-pNA chromogenic assay before committing the protein to downstream experiments.