Serpin B-1 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant Serpin B-1 on SDS-PAGE or Western blot?

Serpin B-1 (SERPINB1, UniProt P30740) is a 379-amino-acid protein with a calculated molecular weight of approximately 42 kDa. On reducing SDS-PAGE, our HEK293-expressed recombinant typically runs at 44–46 kDa — the slight upward shift relative to the calculated mass is common for serpins and reflects the compact, globular fold rather than any glycosylation event (SERPINB1 is not a glycoprotein). Purity is >95% by SDS-PAGE. A clean single band at this position is the expected result under standard reducing conditions.

Which proteases does recombinant Serpin B-1 inhibit and what is its mechanism of action?

Serpin B-1 functions as a suicide-substrate inhibitor — it forms a covalent, SDS-stable complex with its target protease via a reactive-centre loop (RCL) mechanism. Validated targets include neutrophil elastase, cathepsin G, proteinase-3, chymase, and kallikrein-3, as well as the lymphocyte granule protease granzyme H (GZMH). It also suppresses CARD-dependent activation of inflammatory caspases CASP1, CASP4, and CASP5. If your experiment involves neutrophil serine protease signalling or inflammasome regulation, confirm that your chosen target is on this list before designing inhibition assays.

What substrate and assay conditions should I use to measure Serpin B-1 inhibitory activity against neutrophil elastase?

A standard approach is a fluorescence-based progress-curve assay using the synthetic substrate MeOSuc-AAPV-AMC (50–200 µM) with human neutrophil elastase (HNE) as the target enzyme in 50 mM HEPES pH 7.4, 150 mM NaCl, 0.01% Tween-20 at 37°C. Pre-incubate Serpin B-1 with HNE for 5–15 minutes before adding substrate, since serpin inhibition is time-dependent. Monitor AMC release at Ex/Em 360/460 nm. Association rate constants (ka) for SERPINB1–HNE are in the range of 10⁶–10⁷ M⁻¹s⁻¹, so even nanomolar concentrations of inhibitor produce measurable suppression within minutes.

What starting concentration of recombinant Serpin B-1 should I use for a neutrophil elastase inhibition assay?

For a standard stoichiometry-of-inhibition (SI) or progress-curve experiment, start with 50–500 nM Serpin B-1 and titrate against a fixed HNE concentration (e.g., 10–50 nM enzyme). Because serpins form 1:1 covalent complexes with a stoichiometric inhibition ratio typically between 1 and 2, you can estimate the active inhibitor fraction from the molar ratio at which full inhibition is achieved. Avoid using molar excess >10-fold over enzyme in initial screens, as non-specific background can obscure the SI calculation. Our recombinant is provided at a defined concentration stated on the vial CoA.

Does the storage buffer for recombinant Serpin B-1 interfere with serine protease activity assays?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with most fluorogenic serine protease assays at standard dilutions of 1:10 or greater into assay buffer. Glycerol at the working concentration (≤1% after dilution) has negligible effect on HNE or cathepsin G activity. Tris can interfere with some colorimetric substrates, so if you are using a pNA-coupled substrate, dilute into HEPES-based assay buffer instead. The buffer contains no EDTA, DTT, or protease inhibitor cocktail components that would confound serpin inhibition readouts.

Can I use recombinant Serpin B-1 as a positive control for Western blot with the matched RP-SerpinB1 antibody?

Yes — this is a validated pairing. The matched rabbit polyclonal antibody RP-SerpinB1 (/anti-serpin-b1-rabbit-polyclonal-antibody) was raised in the same laboratory that produced this recombinant, ensuring antigen–antibody compatibility. Load 20–50 ng of recombinant protein per lane alongside your cell lysate lanes. Expect a single band at 44–46 kDa under reducing conditions. This setup provides a reliable size and signal reference when validating RP-SerpinB1 on new cell or tissue lysates, and is particularly useful when working with samples where endogenous SERPINB1 expression is low or variable (e.g., non-neutrophil lines).

How much recombinant Serpin B-1 should I load as a Western blot positive control to avoid signal saturation?

In our hands, 20–50 ng per lane gives a clean, quantifiable band with RP-SerpinB1 at antibody dilutions of 1:1,000–1:5,000 using standard HRP-conjugated secondaries and chemiluminescent detection. If you are using fluorescent secondary detection, 50–100 ng may be preferable to remain in the linear dynamic range of your imager. Run a brief titration (10, 25, 50, 100 ng) on the first use with a new lot of RP-SerpinB1, as secondary antibody and detection system variables affect optimal loading more than the primary antibody in most WB workflows.

How should I handle, dilute, and store recombinant Serpin B-1 to maintain activity over time?

Serpin B-1 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol in single-use aliquots and should be stored at -20°C. Avoid repeated freeze-thaw cycles, as each cycle can reduce the fraction of correctly folded, inhibition-competent serpin. Upon first thaw, briefly centrifuge the vial (1,000 × g, 30 s) to collect any condensate, then keep on ice during use. For dilutions below ~100 nM, add BSA (0.1 mg/mL) to the working buffer to prevent adsorptive losses to tube walls. Working aliquots stored at 4°C are stable for up to 48 hours; re-freeze unused material only if aliquot design permits single use.