Serpin A-1 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Serpin A-1 run at on SDS-PAGE or Western blot?

The full-length human Serpin A-1 (418 aa) has a predicted polypeptide mass of approximately 46.7 kDa, but this recombinant is produced in HEK293 cells and carries native-like N-linked glycosylation at four sites (Asn-46, Asn-83, Asn-247, Asn-303). As a result, the apparent molecular weight on a reducing SDS-PAGE or Western blot typically resolves between 52–60 kDa, consistent with the fully glycosylated circulating form. Variation within that range is normal and depends on gel percentage and running conditions. A 10–12% acrylamide gel gives the cleanest separation from contaminating bands.

Is this recombinant Serpin A-1 the native secreted form or an intracellular isoform?

This recombinant corresponds to the canonical secreted isoform (UniProt P01009-1), which includes the 24-amino-acid signal peptide that is co-translationally cleaved during processing in HEK293 cells. The final expressed protein therefore begins at Glu-25 and spans through Lys-418, matching the mature circulating plasma form. Researchers studying the intracellular or Z-variant (Glu342Lys) forms of SERPINA1 should note that this product does not represent those species; it is the wild-type, secreted conformation and is suitable as a reference standard for plasma-derived comparisons.

What does recombinant Serpin A-1 inhibit and what is its primary protease substrate?

Serpin A-1's principal target is human neutrophil elastase (HNE), which it inhibits irreversibly via a conformational trapping mechanism — the reactive center loop (RCL) acts as bait, and cleavage by elastase triggers a large conformational change that covalently traps the protease. Beyond HNE, this recombinant shows measurable inhibitory activity against chymotrypsin, plasmin, thrombin, and plasminogen activators, making it useful for serine protease inhibition panels. For activity confirmation, pairing with recombinant HNE and a fluorogenic substrate such as MeOSuc-AAPV-AMC (HNE-preferred) is the most straightforward approach.

What activity assay and fluorogenic substrate should I use to confirm Serpin A-1 inhibitory activity?

The most widely validated substrate for confirming Serpin A-1 activity against neutrophil elastase is MeOSuc-Ala-Ala-Pro-Val-AMC (MeOSuc-AAPV-AMC), typically used at 200–400 µM in 50 mM HEPES pH 7.4, 150 mM NaCl, 0.01% Tween-20 at 37°C. Pre-incubate Serpin A-1 with HNE (molar ratio 2:1 to 5:1 inhibitor:protease) for 10–15 minutes before adding substrate. AMC fluorescence (Ex/Em 360/460 nm) should show dose-dependent suppression relative to a no-inhibitor control. A stoichiometric inhibition constant (SI) of approximately 1.0–1.2 is expected for native-like preparations under these conditions.

What buffer is Serpin A-1 supplied in and can I dilute it directly into my assay buffer?

This recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For most serine protease inhibition assays — commonly run in HEPES- or PBS-based buffers at neutral pH — direct dilution into assay buffer is compatible provided glycerol carryover stays below 0.5% v/v in the final reaction volume, as higher glycerol can modestly reduce protease activity. If glycerol is a concern, buffer exchange via a 10 kDa spin column is straightforward given the protein's size. Avoid buffers containing strong reducing agents (e.g., >1 mM DTT), which can disrupt the native conformation.

What starting concentration of recombinant Serpin A-1 should I use in an elastase inhibition assay?

For a dose-response inhibition curve, a practical starting point is 500 nM Serpin A-1, titrated down in 2- to 3-fold steps to approximately 10 nM, with HNE held constant at 100–200 nM. At a 2:1 to 5:1 molar excess of Serpin A-1 to HNE, you should see near-complete suppression of AMC fluorescence. Because serpin inhibition is stoichiometric rather than purely competitive, classical IC50 interpretation differs from small-molecule inhibitors — report the apparent SI and effective inhibitory concentration rather than a simple IC50 where possible.

Can I use recombinant Serpin A-1 as a Western blot positive control with the RP-SerpinA1 antibody?

Yes — that is a primary intended use case. This recombinant (REC-SerpinA1) and the matched rabbit polyclonal antibody (RP-SerpinA1) are produced and validated in the same laboratory, so compatibility on Western blot is confirmed rather than assumed. Load 50–100 ng of recombinant protein per lane on a 10–12% SDS-PAGE gel under reducing conditions; the antibody should detect a clean band at 52–60 kDa (glycosylated form). This also serves as a reliable band-size reference when probing tissue lysates that may contain the deglycosylated (~47 kDa) or complexed forms alongside the mature secreted protein.

How should I store recombinant Serpin A-1 and what is its shelf life after aliquoting?

Store at -20°C in the single-use aliquots provided — repeated freeze-thaw cycles progressively reduce inhibitory activity and can promote serpin polymerization, which is particularly relevant for this protein family. Working aliquots are stable at 4°C for up to 72 hours if kept in storage buffer with the 10% glycerol cryoprotectant intact. For long-term storage beyond 12 months, -80°C is preferable. Do not dilute into carrier-free PBS before freezing; the glycerol in the supplied buffer is essential for maintaining the native, active conformation during freeze storage.