Serpin A-3 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Serpin A-3 on SDS-PAGE and Western blot?

The full-length human Serpin A-3 polypeptide has a calculated molecular weight of ~47 kDa based on its 423-amino-acid sequence (UniProt P01011). Because this recombinant is expressed in HEK293 cells, N-linked glycosylation adds approximately 10–20 kDa, so the predominant band typically resolves at 55–68 kDa on reducing SDS-PAGE. Run alongside a broad-range ladder and do not be alarmed by smearing near the upper edge of that range — it reflects heterogeneous glycosylation, not degradation. Under denaturing conditions, a single diffuse band in that window is the expected result.

Does recombinant Serpin A-3 retain the native signal peptide and is it full-length or processed?

The secreted, mature form of human Serpin A-3 begins at residue 25 following cleavage of the 24-residue signal peptide. This recombinant is produced as the mature secreted form (residues 25–423), reflecting the species found in plasma and tissue fluid. No additional pro-peptide processing is required for inhibitory activity — the reactive center loop (RCL) is intact and accessible. The HEK293 expression system ensures the correct disulfide environment and glycosylation pattern, both of which influence RCL conformation and target-protease engagement.

What proteases does recombinant Serpin A-3 inhibit and what enzymatic substrates can I use to measure inhibition?

Serpin A-3 is a canonical inhibitor of neutrophil cathepsin G and mast cell chymase, both chymotrypsin-like serine proteases that cleave after bulky hydrophobic residues. For inhibition-kinetics assays, fluorogenic substrates such as Suc-AAPF-AMC (cathepsin G) or Suc-LLVY-AMC (chymase) at 100–200 µM in 50 mM Tris-HCl pH 7.5, 150 mM NaCl work well. Pre-incubate Serpin A-3 with the target protease for 10–15 minutes at 37°C before substrate addition to allow stoichiometric complex formation. Serpin-protease inhibition is irreversible; IC50 measurements must account for the time-dependent nature of this mechanism.

What starting concentration of recombinant Serpin A-3 should I use for cathepsin G inhibition assays?

For substrate-competition and inhibition-kinetics experiments, we recommend titrating Serpin A-3 across a 10–500 nM range against a fixed cathepsin G concentration of 10–50 nM. Because serpins act as suicide substrates and form a covalent acyl-enzyme intermediate, the stoichiometry of inhibition (SI) must be determined empirically — typically SI = 1–3 for Serpin A-3 / cathepsin G under physiological ionic conditions. Use the supplied storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) as the diluent, and confirm protein concentration by A280 or BCA before each experiment.

Can I use recombinant Serpin A-3 as a positive control for Western blot with the matched TPB antibody RP-SerpinA3?

Yes — this is one of the primary intended uses. REC-SerpinA3 and RP-SerpinA3 are produced and validated in the same lab, so compatibility is confirmed rather than assumed. Load 20–50 ng of REC-SerpinA3 per lane on a 10–12% SDS-PAGE gel; the antibody detects the glycosylated band at 55–68 kDa under reducing conditions. RP-SerpinA3 is a rabbit polyclonal validated for Western blot. Using the recombinant as a positive control alongside your cell lysate or plasma sample removes ambiguity about whether a negative result reflects absent target or antibody failure.

How much recombinant Serpin A-3 should I load for a Western blot positive control lane?

20–50 ng per lane is sufficient for a clean positive control band using RP-SerpinA3 at a 1:1,000–1:2,000 dilution with standard ECL detection. At 20 ng you will see a clear band without overwhelming neighboring sample lanes; scale up to 50 ng if your ECL exposure windows are short or if you are titrating antibody concentration. Prepare a working dilution of REC-SerpinA3 in 1× Laemmli sample buffer and boil for 5 minutes. The resolved band at 55–68 kDa confirms both antibody functionality and correct molecular weight expectation for endogenous Serpin A-3 in your experimental samples.

What buffer conditions are optimal for Serpin A-3 activity assays and does glycerol interfere?

Serpin A-3 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For most activity assays, diluting 10-fold or more into assay buffer reduces glycerol to ≤1%, which does not meaningfully affect cathepsin G or chymase kinetics. Avoid PBS without supplemental NaCl — ionic strength below 100 mM can destabilize the RCL conformation. If your assay requires detergent (e.g., 0.01% Tween-20 to reduce surface adhesion), confirm that total detergent remains below the CMC. pH should be held at 7.0–7.6; Serpin A-3 inhibitory activity drops measurably below pH 6.5.

How should I store and handle recombinant Serpin A-3 to preserve activity over time?

Store at -20°C in the single-use aliquots as supplied. Repeated freeze-thaw cycles progressively denature the RCL and reduce inhibitory stoichiometry — even two additional cycles can produce a measurable increase in SI. On the day of use, thaw one aliquot on ice, spin briefly at 10,000 × g to pellet any microaggregates, and keep on ice throughout the experiment. Shelf life is 12 months from the date of receipt under proper storage. If you anticipate frequent use, prepare working aliquots of 5–10 µg in the supplied buffer and store those at -80°C, which provides greater stability than -20°C for long-term archiving.