Serpin B-13 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Serpin B-13 on SDS-PAGE or Western blot?

Recombinant Serpin B-13 (REC-SerpinB13) is the full-length human protein, residues 1–391, expressed in HEK293 cells. The predicted molecular weight based on amino acid sequence is approximately 42 kDa, but the apparent MW on reducing SDS-PAGE typically runs at 44–46 kDa due to mammalian post-translational modifications. No signal peptide is cleaved (Serpin B-13 is an intracellular clade B serpin), so you should not expect a processed or secreted form. Purity is >90% by SDS-PAGE; a single dominant band at this position confirms intact protein.

Does recombinant Serpin B-13 undergo any post-translational processing or exist as multiple isoforms?

Serpin B-13 (UniProt Q9UIV8) is encoded as a single 391-residue polypeptide with no confirmed signal peptide or propeptide cleavage. Unlike secreted serpins, it lacks a canonical N-terminal secretory sequence, consistent with its intracellular cytoplasmic localization. HEK293 expression provides mammalian glycosylation and folding machinery, which is relevant for conformation-dependent interactions. No alternative isoforms arising from differential splicing of SERPINB13 are currently annotated in UniProt. What you receive is full-length, single-form protein — useful when conformational integrity of the reactive center loop (RCL) matters for your assay.

What proteases does Serpin B-13 inhibit and which enzymes should I use in an activity assay?

Serpin B-13 has been reported to inhibit cysteine proteases, notably cathepsin L and, to a lesser extent, cathepsin K — atypical targets for a serine protease inhibitor family member, placing it among the cross-class inhibitory serpins. For a standard inhibition assay, pair REC-SerpinB13 with recombinant cathepsin L using a fluorogenic substrate such as Z-Phe-Arg-AMC (10–50 µM) in 50 mM sodium acetate, 4 mM EDTA, 8 mM DTT, pH 5.5. Measure fluorescence at Ex/Em 355/460 nm. Note that the standard TPB storage buffer (50 mM Tris-HCl pH 7.5) should be diluted or exchanged prior to acidic-pH cathepsin assays.

What starting concentration of recombinant Serpin B-13 should I use for a protease inhibition assay?

For inhibition assays with cathepsin L, a practical starting range for REC-SerpinB13 is 50–500 nM, used against 1–5 nM active cathepsin L under your chosen substrate conditions. Serpin–protease interactions are stoichiometric (1:1 covalent acyl-enzyme intermediate) rather than purely competitive, so IC50 measurements are less informative than SI (stoichiometry of inhibition) determinations. Titrate Serpin B-13 at 5–10 molar equivalents above the protease concentration as a starting point. Confirm activity by residual protease fluorescence signal dropping >70% at saturating inhibitor concentrations. Pilot experiments in triplicate will establish your specific SI value.

Can I use recombinant Serpin B-13 as a positive control for Western blot with the matched TPB antibody RP-SerpinB13?

Yes — this is one of the most straightforward uses of REC-SerpinB13. The matched rabbit polyclonal antibody RP-SerpinB13 (/anti-serpin-b13-rabbit-polyclonal-antibody) is produced and validated in the same laboratory against the same protein, guaranteeing band identity. Load 20–50 ng of REC-SerpinB13 per lane alongside your cell lysate samples. Expect a clean band at ~44–46 kDa under reducing conditions. This loading range gives strong signal without overwhelming the detection system at standard antibody dilutions. Using this recombinant as your positive control also validates antibody lot-to-lot consistency across experiments.

How much recombinant Serpin B-13 should I load for Western blot positive control and what antibody dilution matches?

Load 20–50 ng of REC-SerpinB13 per lane for Western blot with RP-SerpinB13. At this range, a 1:1,000–1:2,000 primary antibody dilution (RP-SerpinB13) typically yields a well-resolved band at ~44–46 kDa with standard HRP-conjugated secondary antibodies and ECL detection. If you are running a high-sensitivity chemiluminescent system, start at 20 ng to avoid signal saturation. Because RP-SerpinB13 is validated rather than merely predicted for Western blot reactivity, band interpretation alongside the recombinant standard removes ambiguity about endogenous band identity in keratinocyte or epithelial cell lysates.

What buffer is recombinant Serpin B-13 supplied in and how should I handle it for long-term storage?

REC-SerpinB13 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — a formulation that stabilizes the native serpin fold without requiring reducing agents or detergents. Store at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can promote serpin polymerization and loss of RCL-dependent inhibitory activity. Do not store at 4°C long-term. For dilutions needed in assay buffers, use a carrier protein such as 0.1% BSA to prevent adsorption losses at low concentrations (<10 nM). Thawed aliquots used within 24 hours at 4°C retain activity reliably in our internal QC testing.

Is recombinant Serpin B-13 suitable for cell-based experiments and what endotoxin level should I expect?

REC-SerpinB13 is tested at <0.1 EU/µg by the LAL (Limulus Amebocyte Lysate) chromogenic assay, making it appropriate for cell-based assays where endotoxin contamination would confound results — particularly relevant given Serpin B-13's reported role in keratinocyte UV-stress responses where inflammatory signaling is a confounder. Note that as an intracellular serpin, exogenous addition to culture medium will not recapitulate physiological localization; consider microinjection or cell-permeabilization approaches if intracellular delivery is needed. Purity is confirmed at >90% by reducing SDS-PAGE, and the HEK293 expression system provides human-compatible post-translational modifications.