Serpin B-9 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Serpin B-9 on SDS-PAGE and Western blot?

Serpin B-9 (UniProt P50453, isoform 1) has a calculated molecular weight of approximately 42 kDa based on its 376-amino-acid sequence. On reducing SDS-PAGE, the recombinant protein produced in HEK293 cells typically resolves as a single band at ~42–44 kDa. The slight upward shift relative to the calculated mass is common for HEK293-expressed serpins and reflects glycosylation or charge contributions. Purity is >95% by SDS-PAGE. If running a Western blot positive control alongside RP-SerpinB9, expect the band between the 37 and 50 kDa markers.

Is this recombinant Serpin B-9 the full-length protein or a truncated active fragment?

This is the full-length 376-residue sequence of human Serpin B-9, corresponding to UniProt P50453 isoform 1, with no truncations or domain deletions. Unlike secreted serpins, Serpin B-9 lacks a signal peptide, and the full-length cytoplasmic form is the biologically relevant inhibitory species. The reactive centre loop (RCL), which presents the P1–P1' residues critical for granzyme B engagement, is intact. HEK293 expression preserves the native tertiary fold required for the stressed (inhibitory) serpin conformation.

What does recombinant Serpin B-9 cleave, and what is its inhibitory target in vitro?

Serpin B-9 does not itself cleave substrates — it is a serine protease inhibitor whose primary documented target is granzyme B (GZMB), a chymotrypsin-like serine protease with a preference for cleavage after Asp residues. In vitro, recombinant Serpin B-9 forms a stable, covalent SDS-resistant complex with granzyme B via its RCL, effectively titrating granzyme B activity. It does not have documented inhibitory activity against granzyme A, elastase, or thrombin at physiologically relevant concentrations, making it a reasonably selective tool for granzyme B inhibition assays.

How do I set up a granzyme B inhibition assay with recombinant Serpin B-9 — what substrate and buffer conditions should I use?

For granzyme B inhibition assays, the standard fluorogenic substrate is Ac-IEPD-AMC (50–100 µM), which is cleaved efficiently by granzyme B at Asp. Run reactions in 50 mM HEPES pH 7.4, 150 mM NaCl, 0.01% Tween-20 at 37°C. Pre-incubate recombinant Serpin B-9 with a fixed concentration of active granzyme B (typically 2–10 nM) for 15–30 minutes before adding substrate to allow stoichiometric complex formation. Because serpins inhibit via a suicide substrate mechanism, the stoichiometry of inhibition (SI) — not a classical IC50 — is the key parameter to determine; expect SI values near 1.0–2.5 for well-folded preparations.

What starting concentration of recombinant Serpin B-9 should I use for a granzyme B inhibition titration?

Begin with a molar titration series spanning 0.5× to 10× the concentration of your granzyme B stock. If working with 5 nM granzyme B, titrate Serpin B-9 from roughly 2.5 nM to 50 nM. Because the inhibition is stoichiometric rather than kinetic in the conventional sense, complete inhibition is observed when Serpin B-9 reaches approximately 1:1 to 2:1 molar excess over granzyme B. The recombinant is supplied at a defined concentration; dilute into the assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, our storage buffer) or the recommended assay buffer without glycerol for activity work.

Can I use recombinant Serpin B-9 as a positive control for Western blot with the RP-SerpinB9 antibody?

Yes — this is a validated use case. Load 20–50 ng of recombinant Serpin B-9 per lane alongside your cell lysate samples. The matched rabbit polyclonal antibody RP-SerpinB9 (/anti-serpin-b9-rabbit-polyclonal-antibody) is raised from the same lab and is guaranteed compatible for Western blot. The recombinant will produce a clean band at ~42–44 kDa, confirming antibody specificity and correct molecular weight assignment in your system. This pairing is particularly useful when validating RP-SerpinB9 in lysates from immune or tumor cell lines where endogenous Serpin B-9 expression is variable.

How much recombinant Serpin B-9 should I load as a Western blot positive control, and what antibody dilution is recommended?

Load 25–50 ng per lane for a strong, clean signal with RP-SerpinB9. At 25 ng you get a band comfortably above background without saturating the signal, which is helpful when running alongside lower-expressing cell lysates. For RP-SerpinB9 antibody dilution on Western blot, refer to the antibody datasheet at /anti-serpin-b9-rabbit-polyclonal-antibody; typical starting dilutions for rabbit polyclonals in this format are 1:500–1:2000. The recombinant and antibody are produced in the same lab (TPB has been making protease/inhibitor antibody pairs since 1994), so lot-to-lot compatibility is consistent.

How should I store and handle recombinant Serpin B-9 to preserve inhibitory activity — can I avoid freeze-thaw cycles?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and shipped on dry ice. Store at -20°C in single-use aliquots; avoid repeated freeze-thaw cycles, which progressively denature the serpin fold and shift the conformational equilibrium toward the latent (inactive) form. If you anticipate using the protein over several days, keep working aliquots at 4°C for up to 48–72 hours. Do not dilute far below 0.1 mg/mL without adding carrier protein (0.1% BSA) to prevent adsorptive loss on tube surfaces. Endotoxin is <0.1 EU/µg by LAL, suitable for cell-based assays.