Elafin (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant Elafin on SDS-PAGE and Western blot?

The mature Elafin sequence (UniProt P19957) encodes a 117-amino-acid protein with a predicted molecular weight of approximately 9.9 kDa. However, HEK293-expressed recombinant Elafin typically migrates at 12–15 kDa under reducing SDS-PAGE conditions due to native-like N-linked glycosylation. Under non-reducing conditions, the four conserved disulfide bonds of the WAP domain can cause aberrant migration; always run reducing gels unless your assay specifically requires non-reduced samples. Purity is confirmed at >95% by SDS-PAGE.

Is this recombinant Elafin the mature secreted form or does it include the transglutaminase-binding trappin domain?

This construct covers the full-length mature protein as annotated in UniProt P19957, which includes both the N-terminal cementoin/trappin domain (responsible for transglutaminase-mediated cross-linking to ECM) and the C-terminal WAP four-disulfide core domain that carries the elastase-inhibitory activity. The 22-amino-acid signal peptide is absent, as expected for the secreted form. Researchers studying tissue-bound Elafin deposition versus free soluble inhibitor should take the presence of the trappin domain into account when designing binding or cross-linking assays.

What substrates and assay conditions should I use to measure recombinant Elafin inhibitory activity against neutrophil elastase?

The standard functional assay pairs recombinant Elafin against human neutrophil elastase (HNE) using the chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (MeOSuc-AAPV-pNA) at 1–2 mM in 100 mM HEPES pH 7.5, 0.05% Tween-20, 37 °C. Pre-incubate Elafin with HNE for 15–30 minutes before substrate addition to allow tight-binding complex formation. Residual elastase activity is read at A405 nm. This assay is also suitable for pancreatic elastase. Elafin should not be used to inhibit other serine proteases such as thrombin or trypsin, where it shows no meaningful inhibition.

What is the expected IC50 of recombinant Elafin against human neutrophil elastase and what starting concentration do you recommend?

Elafin is a tight-binding, near-stoichiometric inhibitor of human neutrophil elastase (Ki reported in the low nanomolar range, ~0.2–1 nM). Because tight-binding kinetics make classical IC50 determination enzyme-concentration-dependent, apparent IC50 values vary with HNE concentration used. For a practical starting point, use a 1:1 to 5:1 molar ratio of Elafin to HNE, beginning at 50–200 nM Elafin with 50 nM HNE. Confirm activity in your specific buffer — the product is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and is compatible with standard elastase assay buffers after dilution.

Does the storage buffer for recombinant Elafin interfere with elastase activity assays?

The supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is generally compatible with elastase inhibition assays when Elafin is diluted at least 1:10 into the assay buffer. Glycerol at >1% final concentration can slightly reduce substrate cleavage rates; we recommend diluting to ≤1% glycerol in the assay. Tris and NaCl at these carry-over concentrations do not meaningfully affect HNE activity. If your assay requires phosphate-buffered conditions, dilute the protein into assay buffer immediately before use — Elafin is stable for up to 24 hours at 4 °C post-dilution.

Can I use recombinant Elafin as a positive control for Western blot with the TPB anti-Elafin rabbit polyclonal antibody?

Yes. Recombinant Elafin (REC-Elafin) is the recommended positive control for the matched rabbit polyclonal antibody (RP-Elafin; see /anti-elafin-rabbit-polyclonal-antibody). Both reagents are produced and validated in the same laboratory, ensuring epitope compatibility. Load 10–50 ng of recombinant Elafin per lane on a 15–18% SDS-PAGE gel (reducing conditions); the antibody detects the glycosylated band at 12–15 kDa. This pairing is also suitable for antibody titration experiments and as a molecular weight reference when probing tissue lysates where endogenous Elafin may appear at the same apparent MW.

How much recombinant Elafin should I load as a Western blot positive control, and what band will I see?

For a clear, non-saturating signal with the RP-Elafin antibody, load 20–50 ng per lane. At 20 ng you should detect a single band at ~12–15 kDa (reducing, denaturing conditions) corresponding to the glycosylated mature protein. If you observe a faint secondary band near 10 kDa in some preparations, this reflects a minor deglycosylated species and is normal. Use a 15% or 4–20% gradient gel to resolve this size range adequately. Endotoxin is <0.1 EU/µg by LAL, so the recombinant is also suitable as a loading control in cell-stimulation experiments where LPS contamination must be excluded.

What is the recommended storage and handling protocol for recombinant Elafin to maintain activity long-term?

Store recombinant Elafin at -20 °C in the single-use aliquots as supplied. Avoid repeated freeze-thaw cycles — each cycle can reduce WAP-domain disulfide integrity and measurably decrease elastase-inhibitory activity. Upon first thaw, briefly centrifuge (1,000 × g, 30 s) to collect any condensate, then dilute to working concentration in assay buffer immediately before use. For short-term use within 48 hours, the protein is stable at 4 °C. Do not store at room temperature. Carrier protein (0.1% BSA) may be added to diluted aliquots if protein loss to tube surfaces is a concern at concentrations below 10 µg/mL.