Serpin A-5 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Serpin A-5 run at on SDS-PAGE or Western blot?

The unglycosylated polypeptide backbone of human Serpin A-5 (SERPINA5) predicts ~45.6 kDa from the 406-amino-acid sequence. Because this lot is expressed in HEK293 cells and carries native N-linked glycosylation, the apparent molecular weight on reducing SDS-PAGE typically runs between 55–65 kDa — consistent with the endogenously secreted plasma form. Under non-reducing conditions, migration may shift slightly. If your gel shows a band in the 55–65 kDa range, that is expected; a ladder-confirmed band outside that window warrants buffer or reducing agent review.

What is the active inhibitory mechanism of Serpin A-5 and which serine proteases does it target?

Serpin A-5 is a suicide-substrate inhibitor: it presents a reactive center loop (RCL) as a pseudo-substrate, allowing the target protease's catalytic serine to form a covalent acyl-enzyme intermediate. This complex is kinetically trapped and irreversible. Documented targets include activated protein C (APC), prothrombin, factor Xa, factor XI, plasma kallikrein, tissue-type plasminogen activator (tPA), and urokinase plasminogen activator (uPA). This broad inhibitory profile spans both coagulation and fibrinolysis, making Serpin A-5 relevant to thrombosis, hemostasis, and reproductive-biology research models.

What activity assay and chromogenic substrate should I use to measure Serpin A-5 inhibitory activity in vitro?

The most straightforward format is a progress-curve inhibition assay using APC or factor Xa as the target protease paired with a matched chromogenic substrate (e.g., Pefachrome APC or S-2222 for fXa, monitored at A405). Pre-incubate Serpin A-5 with the target protease at a 2–10-fold molar excess over enzyme for 15–30 min at 37 °C in 50 mM Tris-HCl pH 7.5, 150 mM NaCl before adding substrate. A dose-dependent reduction in residual protease activity confirms inhibitory function. Include a protease-only control and a heat-inactivated Serpin A-5 negative control.

What starting concentration and buffer conditions are recommended for Serpin A-5 inhibition assays?

A working concentration of 100–500 nM Serpin A-5 is a reasonable starting range for kinetic inhibition assays against APC or fXa (typically used at 1–5 nM enzyme). The supplied storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with most chromogenic assay systems; dilute into assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% BSA) to minimize surface adsorption. Avoid calcium-chelating additives such as EDTA, which can interfere with coagulation-factor cofactor requirements in complex cascade assays.

How does HEK293 expression affect Serpin A-5 glycosylation and inhibitory activity compared to E. coli-expressed versions?

SERPINA5 carries N-linked glycosylation sites that are absent in bacterially expressed protein. Glycosylation contributes to conformational stability of the native serpin fold — including the metastable stressed conformation that is required for RCL-mediated protease trapping. HEK293-derived Serpin A-5 from this lot retains glycosylation patterns consistent with the secreted plasma form, which supports correct RCL presentation and inhibitory stoichiometry. E. coli-expressed Serpin A-5 frequently requires refolding and may show partial or aberrant inhibitory activity; direct comparison in your assay format is advisable before substituting sources.

Can I use recombinant Serpin A-5 as a positive control on Western blot with the matched antibody RP-SerpinA5?

Yes — this is a validated use case. Load 20–50 ng of recombinant Serpin A-5 (REC-SerpinA5) per lane on a 10–12% SDS-PAGE gel under reducing conditions. The matched rabbit polyclonal antibody RP-SerpinA5 (/anti-serpin-a5-rabbit-polyclonal-antibody) was raised in the same pipeline against this protein and is confirmed compatible for Western blot. Expect a prominent band at 55–65 kDa. This pairing is particularly useful for validating antibody lot-to-lot consistency and for establishing a reference band alongside plasma or cell lysate samples in coagulation or reproductive biology studies.

How much recombinant Serpin A-5 should I load for a Western blot positive control alongside cell lysates?

20–50 ng per lane is the recommended range when using RP-SerpinA5 at a primary antibody dilution of 1:1,000–1:5,000. At 20 ng, the band at ~55–65 kDa is clearly visible with standard ECL or fluorescent secondary detection without overwhelming adjacent lysate lanes. If your lysate contains high endogenous SERPINA5 (e.g., liver-derived cell lines or plasma-conditioned media), loading 20 ng of recombinant protein keeps the positive control from obscuring the lysate signal. Titrate the first time to calibrate signal intensity on your specific membrane and detection system.

What are the storage and handling recommendations for recombinant Serpin A-5 to maintain inhibitory activity?

Store at -20 °C in single-use aliquots. The supplied buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — stabilizes the protein during freeze-thaw, but repeated freeze-thaw cycles progressively reduce inhibitory activity by promoting serpin polymer formation or latent-form transition. Once thawed, keep on ice and use within the same working session. Do not dilute into water alone; maintain at least 50 mM Tris-HCl pH 7.4–7.6 with 0.1% BSA as carrier to prevent adsorptive loss at low concentrations (<50 nM). Purity is ≥90% by SDS-PAGE; endotoxin is <0.1 EU/µg by LAL.