Elastase-1 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Elastase-1 on SDS-PAGE and Western blot?

The full-length human Elastase-1 (CELA2A, UniProt P08217) is 269 amino acids, giving a theoretical MW of ~29 kDa. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation and disulfide-bond formation are preserved, so the apparent MW on reducing SDS-PAGE typically runs at 30–33 kDa — slightly higher than the sequence-predicted value. Under non-reducing conditions, expect a band at a similar position since the disulfide bonds are intramolecular. Lot-specific SDS-PAGE images are included in each Certificate of Analysis.

Is recombinant Elastase-1 expressed as the zymogen or the mature active form?

The recombinant is produced as the mature, processed enzyme, not the full zymogen precursor. HEK293 expression supports the secretory processing pathway, and the product is confirmed active upon QC release. The propeptide (signal and activation peptide) is removed during production, yielding the catalytically competent form classified under EC 3.4.21.71. If your experiment requires the zymogen form for processing studies, this product is not appropriate — it is specifically intended for activity-based assays where a folded, active serine protease is needed from the outset.

What substrates does Elastase-1 cleave, and which synthetic peptide substrate works best for activity assays?

Elastase-1 is a chymotrypsin-like serine protease that cleaves at the C-terminal side of small, uncharged residues — primarily Ala, Val, and Ser. For kinetic assays (Km, kcat determination), the chromogenic substrate Suc-Ala-Ala-Ala-pNA (succinyl-tri-alanine p-nitroanilide) is widely used and provides a robust absorbance readout at 405 nm. Fluorogenic substrates such as MeO-Suc-Ala-Ala-Pro-Val-AMC offer higher sensitivity for low-enzyme-concentration or inhibitor-screening formats. Solid-phase elastin (FITC-elastin) is also compatible for bulk elastolytic activity measurements. We recommend starting activity validation before committing the recombinant to a full inhibitor screen.

What buffer conditions should I use for Elastase-1 activity assays, and is the storage buffer compatible?

Elastase-1 is most active in the range of pH 7.5–8.5. A standard assay buffer of 50 mM Tris-HCl pH 8.0, 150 mM NaCl works well for both chromogenic and fluorogenic substrate formats. The product is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — the glycerol must be diluted to ≤1% in final assay volume to avoid substrate solubility issues and fluorescence quenching. Avoid EDTA and reducing agents such as DTT or TCEP at concentrations above 1 mM, as disulfide bond reduction will compromise activity. Pre-warm the enzyme to room temperature on ice for 10 minutes before use.

What starting concentration of recombinant Elastase-1 should I use for IC50 determination against alpha-1 antitrypsin?

For serpin-class inhibitor IC50 assays, including alpha-1 antitrypsin (AAT) titrations, a typical starting enzyme concentration is 1–5 nM in the final assay volume — well below the Km for your chosen substrate so that the Morrison tight-binding correction is not required unless you are working with very potent inhibitors (Ki < 1 nM). With Suc-Ala-Ala-Ala-pNA, use the substrate at or below 200 µM and keep DMSO from inhibitor stocks below 0.5% v/v. Run a no-inhibitor control and a substrate-only blank in every plate. Pre-incubate enzyme with inhibitor for 30 minutes at 37°C before substrate addition to allow full complex formation.

Can I use recombinant Elastase-1 as a positive control for Western blot with the matched anti-Elastase-1 antibody RP-Elastase1?

Yes — this is one of the primary intended uses. The matched rabbit polyclonal antibody RP-Elastase1 (/anti-elastase-1-rabbit-polyclonal-antibody) is raised and validated against Elastase-1 in the same TPB laboratory, ensuring lot-to-lot compatibility. Loading 20–50 ng of recombinant Elastase-1 per lane on a 12% or 4–20% gradient SDS-PAGE gel, under reducing conditions, gives a clean band at ~30–33 kDa that is reliably detected with RP-Elastase1. This recombinant-antibody pair is particularly useful when validating antibody performance on a new tissue lysate or establishing that a band in your sample co-migrates with the authentic protein.

How much recombinant Elastase-1 should I load for a Western blot positive control, and what antibody dilution is recommended?

Load 20–50 ng per lane for a strong, clean signal. At 20 ng you should detect a sharp band at ~30–33 kDa with RP-Elastase1 used at 1:1,000–1:2,000 dilution in 5% non-fat dry milk/TBST with an overnight 4°C incubation. If your tissue or cell lysate lanes are being probed simultaneously, match the positive-control lane to the same gel to avoid inter-blot variability. Avoid loading more than 100 ng — overloaded recombinant lanes can bleed across adjacent sample lanes. HRP-conjugated anti-rabbit secondary at 1:5,000–1:10,000 for 1 hour at room temperature is sufficient for ECL detection.

How should I store and handle recombinant Elastase-1 to maintain activity over time?

Upon receipt, spin down briefly and store at -20°C in single-use aliquots — the supplied format with 10% glycerol is stable for at least 12 months under these conditions. Avoid repeated freeze-thaw cycles; each cycle can result in measurable loss of specific activity for serine proteases. For daily bench use, thaw one aliquot on ice and keep on ice throughout; do not leave at room temperature for extended periods. If you anticipate using the enzyme across multiple days, prepare working dilutions in assay buffer containing 0.1% BSA to stabilize the protein, aliquot these secondary stocks, and store at -80°C for up to 1 month. Endotoxin is <0.1 EU/µg by LAL, making the product suitable for cell-based assays.