Serpin B-10 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Serpin B-10 on SDS-PAGE and Western blot?

Serpin B-10 is a 397-amino-acid protein with a calculated molecular weight of approximately 44.6 kDa. On reducing SDS-PAGE, the recombinant produced in HEK293 cells typically migrates at 45–48 kDa; the slight upward shift relative to the theoretical mass is consistent with glycosylation and other post-translational modifications supported by the mammalian expression system. On Western blot, expect a single predominant band in that same 45–48 kDa range. Anomalous migration is not commonly reported for this serpin.

Is recombinant Serpin B-10 the full-length protein or a processed fragment? Does it undergo autocleavage?

This recombinant corresponds to the full-length 397-residue human Serpin B-10 sequence (UniProt P48595, isoform 1). As a clade B ov-serpin, Serpin B-10 does not possess a signal peptide and is not subject to the secretory processing seen in clade A serpins. Autocleavage at the reactive centre loop (RCL) can occur if the protein is incubated with a cognate protease that cleaves without forming a stable inhibitory complex; under normal storage conditions (no protease present), the intact inhibitory form is maintained. Verify integrity by SDS-PAGE before each experiment.

What protease does Serpin B-10 inhibit and what substrate or protease should I use to measure its inhibitory activity in vitro?

Serpin B-10 (Bomapin) has been reported to inhibit serine proteases, with tryptase and certain cathepsin G-related activities cited in the literature as candidate targets. For in vitro activity assays, a standard approach is to pre-incubate recombinant Serpin B-10 with the target serine protease (e.g., tryptase β1) for 15–30 min at 37 °C in assay buffer, then add a fluorogenic substrate such as Boc-Phe-Ser-Arg-AMC and measure fluorescence at Ex/Em 380/460 nm. Serpin B-10 acts as a stoichiometric inhibitor, so titrate the protease:inhibitor molar ratio (typically 1:1 to 1:5) rather than deriving a classical enzyme IC50.

What buffer conditions are recommended for Serpin B-10 protease inhibition assays and is the storage buffer compatible?

The product is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. This buffer is compatible with most serine protease activity assays, though glycerol carryover above ~2% can affect some fluorescent readouts. For sensitive fluorogenic assays, dilute the recombinant at least 1:5 into assay buffer (e.g., 50 mM HEPES pH 7.4, 150 mM NaCl, 0.01% Tween-20) before use. Avoid reducing agents such as DTT above 1 mM, as high concentrations can destabilize the RCL conformation critical for inhibitory activity. Working pH range 6.8–8.0 is generally well-tolerated.

What starting concentration of recombinant Serpin B-10 should I use in a stoichiometric inhibition assay?

Because serpins act as suicide substrate inhibitors rather than catalytic inhibitors, dosing is expressed as a molar ratio relative to the target protease rather than as an IC50. A practical starting point is 200–500 nM recombinant Serpin B-10 paired with 50–100 nM protease, giving a 2–10 fold molar excess of inhibitor. Confirm complete inhibition by residual substrate cleavage and titrate downward to determine the stoichiometry of inhibition (SI) value for your specific protease preparation. Purity is >90–95% by SDS-PAGE, so mass-based concentration estimates closely reflect active protein, though functional titration against a known protease standard is still advised.

Can I use recombinant Serpin B-10 as a positive control on Western blot with the matched TPB antibody RP-SerpinB10?

Yes — this is one of the primary intended uses of REC-SerpinB10 alongside the matched rabbit polyclonal RP-SerpinB10 (/anti-serpin-b10-rabbit-polyclonal-antibody). The antibody and recombinant are produced and validated in the same laboratory, ensuring epitope compatibility. Load 20–50 ng of recombinant per lane on a 10–12% SDS-PAGE gel; this typically yields a clean, well-resolved band at 45–48 kDa that serves as an unambiguous size reference when probing cell or tissue lysates. RP-SerpinB10 is validated for Western blot; the recombinant positive control is particularly useful when working with sample types where endogenous Serpin B-10 expression is low or uncertain.

How much recombinant Serpin B-10 should I load for Western blot positive control and what band should I expect?

Load 20–50 ng per lane when using RP-SerpinB10 at standard working dilutions (typically 1:500–1:2,000 for Western blot; confirm with the antibody datasheet). At 50 ng with a sensitive HRP-chemiluminescence detection system, the band at 45–48 kDa is clearly visible without background interference. If your endogenous lysate lanes contain high total protein (20–40 µg), run the recombinant in a flanking lane rather than mixing, to avoid signal saturation. The HEK293 expression system means the recombinant band migration closely mirrors endogenous human Serpin B-10, minimising ambiguity in band assignment.

How should I handle, dilute, and store recombinant Serpin B-10 to maintain activity and avoid freeze-thaw degradation?

REC-SerpinB10 is shipped and stored at -20 °C in single-use aliquots in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. Thaw on ice, mix gently by pipetting — do not vortex — and use within the same working session. The 10% glycerol in the storage buffer provides cryoprotection; repeated freeze-thaw cycles progressively denature the RCL and reduce inhibitory activity. If a stock will be used across multiple days, prepare working aliquots of the diluted protein in assay buffer supplemented with 0.1% BSA and store at 4 °C for no more than 48 hours. Do not re-freeze thawed material.