Serpin B-7 (Recombinant)

Frequently Asked Questions

What molecular weight should recombinant Serpin B-7 run at on SDS-PAGE or Western blot?

The full-length 380-amino-acid Serpin B-7 construct (UniProt O75635) has a calculated molecular weight of approximately 42 kDa. On reducing SDS-PAGE, expect a band in the 42–45 kDa range; minor upward shift relative to the predicted MW is common with HEK293-expressed proteins due to glycosylation or other post-translational modifications. Under non-reducing conditions, the apparent MW may shift slightly. Purity is confirmed >95% by SDS-PAGE prior to release. If your observed band deviates significantly from this range, contact us with your gel conditions.

Is recombinant Serpin B-7 full-length or a truncated fragment, and does it retain inhibitory activity?

This construct covers the complete 380-residue sequence of human Serpin B-7 (SERPINB7), expressed in HEK293 mammalian cells to preserve native eukaryotic folding. The reactive center loop (RCL), which is essential for serine protease inhibitory conformation, is intact. Production in HEK293 rather than E. coli is specifically chosen to support correct disulfide environment and folding relevant to inhibitory activity assays. This is the full-length isoform; no truncation or signal peptide clipping is expected for this intracellular clade B serpin.

What protease does Serpin B-7 inhibit, and what substrate or enzyme should I use in an activity assay?

Serpin B-7 (Megsin) has been characterized as an inhibitor of Lys-specific serine proteases. For residual-activity assays, plasmin is a commonly used model enzyme partner given its Lys-preferring cleavage specificity. A fluorogenic substrate such as H-D-Val-Leu-Lys-AMC or the chromogenic H-D-Ala-Lys-pNA can be used to monitor residual protease activity after incubation with Serpin B-7. Pre-incubate the serpin with the target protease (molar ratios of 2:1 to 10:1 serpin:protease) in assay buffer before adding substrate, then measure inhibition relative to a no-serpin control.

What buffer conditions are recommended for Serpin B-7 inhibitory activity assays, and is the storage buffer compatible?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with most serpin activity assays at typical working dilutions (1:5 or greater). For sensitive fluorogenic assays, dilute into your assay buffer (e.g., 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.01% Tween-20) to bring glycerol below 2%, which can otherwise reduce enzyme velocity. Avoid EDTA if your target protease is calcium-dependent. pH 7.4–7.5 is appropriate for preserving the inhibitory conformation of clade B serpins.

What starting concentration of recombinant Serpin B-7 should I use for IC50 or stoichiometry of inhibition experiments?

For stoichiometry-of-inhibition (SI) experiments, titrate Serpin B-7 at molar ratios of 1:1 to 20:1 against your target protease, keeping protease concentration fixed (typically 1–10 nM for sensitive fluorogenic readouts). For IC50 determinations in competition assays, a working range of 10–1000 nM Serpin B-7 is a practical starting point. Because clade B serpins can behave as suicide inhibitors with SI values >1, include a substrate-competition control to distinguish true inhibition from stoichiometric trapping. Protein concentration is provided on the vial Certificate of Analysis.

Can I use recombinant Serpin B-7 as a positive control for Western blot with the matched TPB anti-Serpin B-7 antibody (RP-SerpinB7)?

Yes — this is a validated use case. The matched rabbit polyclonal antibody RP-SerpinB7 (/anti-serpin-b7-rabbit-polyclonal-antibody) was raised against Serpin B-7 and is confirmed compatible with this recombinant protein on Western blot. Load 10–50 ng of recombinant Serpin B-7 per lane alongside your cell lysate samples. Expect a clean band at approximately 42–45 kDa. This pairing is particularly useful for confirming antibody performance and establishing a reliable size reference when probing endogenous Serpin B-7 in megakaryocyte or keratinocyte lysates.

How much recombinant Serpin B-7 should I load for a Western blot positive control lane?

A loading range of 10–50 ng per lane is sufficient for a strong positive control signal when using the matched RP-SerpinB7 rabbit polyclonal antibody. Start with 25 ng to establish baseline signal, then titrate down to 10 ng if background is an issue or up to 50 ng if signal is weak with your secondary antibody system. Run on a 10–12% SDS-PAGE gel for optimal resolution in the 40–50 kDa range. Ensure the lane is not overloaded, as serpin proteins at high concentration can appear as a doublet due to native vs. cleaved (relaxed) conformer migration differences.

How should I store and handle recombinant Serpin B-7 to maintain activity, and what is the shelf life?

Store at -20°C in single-use aliquots upon receipt. 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 should be avoided as they accelerate conformational drift in serpins and can populate the latent (inactive) conformation. Shelf life is 12 months from date of receipt when stored correctly. After thawing, keep on ice and use within 4–8 hours. Do not dilute to below approximately 0.1 mg/mL in plain buffer without adding a carrier protein (e.g., 0.1% BSA) to prevent adsorption losses.