Serpin B-2 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant Serpin B-2 on SDS-PAGE and Western blot?

Recombinant Serpin B-2 (PAI-2; P05120) runs at approximately 47–50 kDa under reducing SDS-PAGE conditions, slightly above its calculated molecular weight of ~45 kDa for the 415-amino-acid sequence. The upward shift is typical for this protein and reflects its glycosylation profile, which is preserved in the HEK293 expression system used here. Under non-reducing conditions a minor high-molecular-weight smear can appear due to intermolecular disulfide-linked species. Purity is >95% by SDS-PAGE. If you are running a Western blot positive control alongside RP-SerpinB2 antibody, load this band as your reference.

Does this recombinant Serpin B-2 correspond to the glycosylated or non-glycosylated isoform of PAI-2?

PAI-2 (SERPINB2) is naturally expressed in two forms: a non-glycosylated intracellular form (~47 kDa) and a secreted, N-glycosylated form (~60 kDa). This recombinant is produced in HEK293 cells with a secretion signal, yielding the glycosylated, secreted-equivalent form that migrates at ~47–50 kDa under our expression conditions. Full 60 kDa glycosylation seen in some primary placental sources is not always recapitulated at equivalent density in HEK293; if your assay is sensitive to glycan composition, PNGase F digestion of a test aliquot before use is advisable for confirmation.

What protease does recombinant Serpin B-2 inhibit and what is the preferred substrate for an activity assay?

Serpin B-2 (PAI-2) is a physiological inhibitor of urokinase-type plasminogen activator (uPA; PLAU). In a functional inhibition assay, incubate recombinant PAI-2 with two-chain uPA, then monitor residual uPA amidolytic activity using the chromogenic substrate Spectrozyme UK (H-D-Glu-Gly-Arg-pNA·2HCl) or the equivalent Boc-Glu-Gly-Arg-AMC fluorogenic substrate at 0.2–0.5 mM in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20. PAI-2 forms a covalent acyl-enzyme complex with uPA's active-site Ser195, providing a clean endpoint for stoichiometric inhibition studies. Tissue-type plasminogen activator (tPA) inhibition is roughly 10-fold weaker and not the recommended primary readout.

What starting concentration of recombinant Serpin B-2 should I use for a uPA inhibition assay and how does IC50 vary with uPA concentration?

Because PAI-2 is a stoichiometric, covalent inhibitor rather than a classical competitive inhibitor, IC50 is not a fixed intrinsic constant — it scales linearly with uPA concentration (a consequence of tight-binding inhibition kinetics). As a practical starting point, use 50–200 nM recombinant PAI-2 paired with 10–50 nM two-chain uPA in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20, 37 °C). Titrate PAI-2 from 0.1× to 5× the uPA molar concentration to bracket the inhibition curve. For second-order rate constant (k2) determinations, pseudo-first-order conditions with a ≥10-fold molar excess of PAI-2 over uPA are standard.

What buffer conditions are optimal for storing and diluting recombinant Serpin B-2 before assay?

The supplied storage buffer is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. Aliquots are stable at −20 °C for at least 12 months; avoid repeated freeze-thaw cycles, as serpins are susceptible to latency conversion (loop insertion without cleavage) upon thermal cycling. For assay dilutions, exchange into or dilute into the same Tris-NaCl buffer minus glycerol, adding 0.01–0.05% Tween-20 or 0.1% BSA as a carrier to prevent surface adsorption at sub-nanomolar concentrations. Do not dilute into phosphate buffers below pH 7.0, as serpin conformation is pH-sensitive in the 6.5–7.0 range.

Can I use recombinant Serpin B-2 as a positive control for Western blot with the RP-SerpinB2 rabbit polyclonal antibody?

Yes — this is the primary intended cross-use case. Recombinant Serpin B-2 (REC-SerpinB2) and the matched rabbit polyclonal antibody (RP-SerpinB2) are developed from the same lab and validated together for Western blot. Load 20–50 ng of recombinant protein per lane alongside your cell lysate lanes; under standard reducing SDS-PAGE you will see a clean ~47–50 kDa band that serves as an unambiguous size and signal reference. The matched antibody page is at /anti-serpin-b2-rabbit-polyclonal-antibody. This pairing is particularly useful when validating antibody lot-to-lot consistency or when establishing detection sensitivity in new cell line lysates.

How much recombinant Serpin B-2 should I load for a Western blot positive control, and what dilution of RP-SerpinB2 antibody should I use?

Load 20–50 ng of REC-SerpinB2 per lane for a positive control band. At this amount, RP-SerpinB2 rabbit polyclonal antibody (SKU: RP-SerpinB2) typically performs well at a primary antibody dilution of 1:1,000–1:2,000 in 5% non-fat milk/TBST with a 1-hour room-temperature or overnight 4 °C incubation. For high-sensitivity detection (e.g., ECL plus or fluorescent secondary), 10–20 ng may be sufficient and will reduce background. Always run the recombinant in a lane separate from the highest-concentration lysate to avoid signal bleed-over distorting band interpretation.

How do I reconstitute or dilute recombinant Serpin B-2 from a frozen stock without losing activity?

Aliquots are supplied ready-to-use in solution (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) — no lyophilization step is required. Thaw a single-use aliquot on ice (10–15 minutes), briefly spin at 10,000 × g for 30 seconds to collect any condensate, then dilute directly into your assay buffer. Avoid vortexing; serpins can partially unfold under shear. If working at concentrations below 10 nM, add 0.1% BSA or 0.05% Tween-20 to prevent adsorptive loss to tube walls. Discard unused diluted material; do not refreeze working dilutions. Opened stock vials can be held at 4 °C for up to 7 days without significant activity loss.