Elastase-2-A (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Elastase-2-A on SDS-PAGE and Western blot?

The full-length human CELA2A protein is 269 amino acids, giving a theoretical MW of ~29 kDa. However, because this recombinant is produced in HEK293 cells, N-linked glycosylation adds approximately 3–6 kDa, so expect a diffuse band running at 32–35 kDa under reducing SDS-PAGE conditions. Non-reducing conditions may shift migration slightly due to intact disulfide bonds. Our matched antibody (RP-Elastase2A) is validated against this glycosylated form, so the band you observe with the recombinant as a positive control should align with endogenous CELA2A in plasma or pancreatic lysates.

Is this recombinant Elastase-2-A the mature processed form or does it include the signal peptide?

The recombinant is expressed as the mature secreted form. The 29-amino-acid signal peptide (residues 1–29) is cleaved co-translationally during secretion from HEK293 cells, so the product begins at the predicted N-terminus of the processed enzyme (Ala-30). The zymogen propeptide is also removed, yielding the catalytically competent serine protease domain. If your experiment requires comparison to a specific isoform or propeptide-containing precursor, confirm the construct boundaries in the Certificate of Analysis before use.

What substrates does recombinant Elastase-2-A cleave and how do I set up a chromogenic activity assay?

CELA2A cleaves the canonical elastase substrate succinyl-Ala-Ala-Ala-p-nitroanilide (Suc-AAA-pNA), with absorbance monitored at 405 nm. A practical starting point: 50–200 nM recombinant Elastase-2-A, substrate concentrations spanning 0.1–5 mM to bracket Km, in 50 mM Tris-HCl pH 7.5, 150 mM NaCl at 37°C. For fluorogenic assays, MeO-Suc-Ala-Ala-Pro-Val-AMC (excitation 380 nm / emission 460 nm) offers greater sensitivity. Run a no-enzyme blank and confirm linearity over at least 10 minutes before extracting initial velocity values for Michaelis-Menten fitting.

What buffer conditions are optimal for Elastase-2-A activity assays and does glycerol in the storage buffer interfere?

Activity is well supported in 50 mM Tris-HCl pH 7.5, 150 mM NaCl — the same buffer used for storage — so minimal buffer exchange is needed. The 10% glycerol in the storage buffer is generally tolerated at final assay concentrations below 1%, achievable by diluting the enzyme at least 1:10 into assay buffer before adding substrate. Avoid EDTA-containing buffers; while CELA2A is a serine protease not requiring divalent cations for catalysis, EDTA can chelate trace metals that stabilise the fold. pH optimum is 7.5–8.0; activity drops noticeably below pH 6.5.

What starting concentration of recombinant Elastase-2-A should I use for inhibitor IC50 screening?

For inhibitor screening, use the minimum enzyme concentration that gives a robust, linear signal — typically 50–100 nM recombinant Elastase-2-A with Suc-AAA-pNA at or below Km (≈0.5 mM is a reasonable estimate for CELA2A). Running enzyme well below substrate Km keeps the competitive inhibition model accurate and avoids substrate depletion artefacts. Pre-incubate enzyme with inhibitor for 15–30 minutes at room temperature before adding substrate to capture slow-binding inhibitors. Always confirm enzyme stability over the incubation period with a no-inhibitor time-course control.

Can I use recombinant Elastase-2-A as a positive control for Western blot with the matched RP-Elastase2A antibody?

Yes — this is one of the primary intended uses. REC-Elastase2A and RP-Elastase2A are produced and validated together in our laboratory, so compatibility is confirmed rather than assumed. Load 20–50 ng of recombinant per lane under reducing conditions; the antibody should detect a clean 32–35 kDa band at typical working dilutions of 1:500–1:2,000 (see the RP-Elastase2A datasheet for the validated dilution range). This positive control is particularly useful when running plasma samples or pancreatic tissue lysates where endogenous CELA2A signal intensity is uncertain.

How much recombinant Elastase-2-A should I load for a Western blot positive control lane?

20–50 ng per lane is the recommended starting point when using RP-Elastase2A at 1:1,000 dilution on standard PVDF or nitrocellulose membranes. If you are optimising a new membrane or detection system, run a titration at 10, 25, and 50 ng to identify the linear detection range before committing to a fixed loading amount. Because the HEK293-expressed protein is glycosylated, it migrates as a slightly diffuse band; overloading (>100 ng) tends to smear rather than sharpen the signal. Pair with a plasma sample of known CELA2A expression to calibrate relative band intensities.

How should I store and handle recombinant Elastase-2-A to preserve enzymatic activity over time?

Store at −20°C in single-use aliquots immediately upon receipt. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is formulated to maintain activity through a single freeze-thaw cycle; repeated freeze-thaw leads to measurable activity loss in serine proteases. Prepare working dilutions fresh in assay buffer on ice and use within 4 hours. If you need to hold diluted enzyme on the bench, add 0.1% BSA (protease-free) as a carrier to reduce adsorptive loss at low concentrations. Do not add reducing agents such as DTT or BME unless your protocol specifically requires them, as these can disrupt the disulfide bonds critical to CELA2A fold integrity.