Serpin B-11 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Serpin B-11 run at on SDS-PAGE or Western blot?

The full-length 392-amino-acid human Serpin B-11 has a predicted molecular weight of approximately 44 kDa. On reducing SDS-PAGE, the recombinant protein (HEK293-expressed) typically migrates between 45–50 kDa — a modest upward shift from the calculated MW that is consistent with glycosylation or other post-translational modifications retained from mammalian expression. When running a Western blot positive control alongside the matched antibody RP-SerpinB11, expect the dominant band in this 45–50 kDa window. Any faint higher-MW band may represent SDS-resistant dimer.

Is Serpin B-11 a processed or cleaved form — does the recombinant cover the full sequence?

Serpin B-11 (UniProt Q96P15) is annotated as a single 392-residue chain with no classical signal peptide and no documented propeptide cleavage, consistent with its clade B (cytoplasmic) classification. This recombinant covers the full 392-residue sequence expressed in HEK293 cells. There are no well-characterised isoforms arising from alternative cleavage in the literature; the intact native-length form is therefore the biologically relevant species for conformation, interaction, and localisation studies. We are not aware of a cleaved active form analogous to the stressed/latent transitions seen in inhibitory serpins.

Does Serpin B-11 inhibit serine proteases — what protease substrates should I use to test activity?

Serpin B-11 is annotated as a non-inhibitory serpin: structural divergence within its reactive-centre loop is thought to prevent the canonical stressed-to-relaxed conformational transition required for covalent serine protease inhibition. Standard chromogenic substrates used for inhibitory serpins (e.g., pNA-conjugated substrates for trypsin, elastase, or thrombin) are not appropriate for functional readout here. Assay design should focus on non-inhibitory functions — protein–protein interaction assays, thermal shift/DSF for conformational stability studies, or cell-based binding assays — rather than classical protease inhibition kinetics.

What buffer conditions are recommended for Serpin B-11 recombinant protein in binding or interaction assays?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — a buffer that supports folded, soluble serpin conformation and is compatible with most pull-down, co-IP, and SPR setups. For interaction assays requiring lower glycerol, the protein can be diluted into assay buffer (PBS pH 7.4 or HEPES-based buffers at comparable ionic strength); keep glycerol ≥2% if the working concentration drops below ~0.1 mg/mL to reduce surface adsorption. Avoid pH below 6.5, as clade B serpins can undergo acid-induced conformational change to the latent form.

What starting concentration should I use for recombinant Serpin B-11 in a protein interaction or thermal shift assay?

For differential scanning fluorimetry (DSF/thermal shift), 2–5 µg per reaction (~0.5–1 µM based on the ~44 kDa MW) is a practical starting point alongside SYPRO Orange at 5× final concentration. For pull-down or co-IP bait loading, 5–10 µg of recombinant Serpin B-11 per condition is typical for a first experiment, titrating prey protein 1:0.5–1:5 molar ratio relative to bait. Purity is >90–95% by SDS-PAGE and endotoxin is <0.1 EU/µg, so cell-based binding assays at 0.1–1 µM extracellular concentrations are feasible without LPS-driven confounds.

Can I use recombinant Serpin B-11 as a positive control for Western blot with the RP-SerpinB11 antibody?

Yes — this is the primary validated use case for pairing REC-SerpinB11 with RP-SerpinB11. The rabbit polyclonal antibody was raised against the same human Serpin B-11 sequence and has been validated by Western blot, so recombinant protein loaded alongside cell lysate provides a defined-MW band at 45–50 kDa for antibody performance confirmation. Load 20–50 ng of recombinant protein per lane; this gives a clean, strong band without saturating the signal at standard ECL exposures. The matched antibody product page (/anti-serpin-b11-rabbit-polyclonal-antibody) lists recommended antibody dilutions for co-use.

How much recombinant Serpin B-11 should I load for antibody validation or positive control on a Western blot?

A titration of 10 ng, 25 ng, and 50 ng per lane is a useful starting range for RP-SerpinB11 antibody validation experiments. At 50 ng you are loading roughly 1 pmol of the ~44 kDa protein, which is well within the linear detection range for most HRP-conjugated secondary antibody and ECL systems. If you are validating alongside endogenous Serpin B-11 in cell lysates — noting that expression appears tissue-restricted — include the recombinant lane as a MW anchor. Avoid loading >200 ng, as excess recombinant serpin can bleed into adjacent lanes on standard mini-gels.

How should I store recombinant Serpin B-11 and what is its shelf life after thawing?

Store at -20°C in the supplied single-use aliquots. Avoid repeated freeze-thaw cycles, as serpins are susceptible to polymerisation under thermal stress — a well-documented artefact for the serpin superfamily. Once thawed, keep on ice and use within 4–8 hours; if same-day use is not possible, the thawed aliquot can be held at 4°C for up to 24 hours without significant aggregation, provided it is not agitated. Unopened aliquots stored continuously at -20°C are stable for at least 12 months from the date of manufacture. Do not dilute into buffer lacking glycerol for long-term storage.