Serpin B-5 (Recombinant)

Frequently Asked Questions

What molecular weight band does recombinant Serpin B-5 / Maspin run at on SDS-PAGE or Western blot?

The predicted molecular weight of full-length Serpin B-5 (375 aa, UniProt P36952) is approximately 42 kDa. Under denaturing SDS-PAGE conditions, our HEK293-expressed recombinant typically migrates at 45–48 kDa — slightly above the theoretical mass due to post-translational modifications supported by the mammalian expression system. On Western blot using the matched antibody (RP-SerpinB5), expect a clean band in the 45–50 kDa range. Anomalous migration by a few kDa is normal and consistent with published endogenous Maspin reports in MCF-7 and LNCaP lysates.

Does recombinant Maspin / Serpin B-5 have serine protease inhibitory activity — can I use it in a protease inhibition assay?

No. Serpin B-5 (Maspin) is a non-inhibitory serpin: it does not undergo the stressed-to-relaxed (S→R) conformational transition required for canonical serine protease inhibition and does not form a covalent acyl-enzyme intermediate with targets such as trypsin, plasmin, or uPA. Do not use this recombinant in standard protease inhibition or IC50 assays expecting reactive centre loop (RCL)-mediated trapping. Its biological activity is attributed to protein–protein interactions (e.g., desmoplakin, TGF-β pathway components) and transcriptional regulatory roles — assay design should reflect those mechanisms.

What assays are appropriate for recombinant Serpin B-5 if it has no protease inhibitory activity?

Validated uses for this recombinant include: (1) protein–protein interaction studies — SPR, co-IP, or pull-down assays probing Maspin's binding to desmoplakin or TGF-β signalling components; (2) Western blot positive control at 50–200 ng per lane using the matched antibody RP-SerpinB5; (3) ELISA standard curve generation for quantifying endogenous Maspin in tissue lysates or conditioned media; and (4) cell-based uptake or localisation experiments where exogenously supplied recombinant protein is tracked. A starting concentration of 1–10 µg/mL is a reasonable working range for most binding assays; titrate to your specific system.

What buffer is recombinant Serpin B-5 supplied in and is it compatible with my binding assay?

REC-SerpinB5 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. This formulation maintains conformational integrity during storage. For SPR or ITC, dialyse or buffer-exchange into your running buffer (e.g., PBS pH 7.4 or HEPES-buffered saline) to avoid refractive index artifacts from glycerol. For co-IP or pull-down, the supplied buffer is generally compatible — simply dilute ≥5-fold into your lysis/binding buffer. Avoid pH extremes (<6.0 or >9.0) and detergent concentrations above 0.1% SDS, which may disrupt the non-inhibitory conformation critical to Maspin's interaction surfaces.

How should I store recombinant Serpin B-5 and what is its shelf life after thawing?

Store at −20°C in single-use aliquots immediately upon receipt. Repeated freeze-thaw cycles accelerate aggregation and should be avoided. Once thawed, keep on ice and use within 8 hours; do not refreeze. Under recommended storage conditions, REC-SerpinB5 is stable for up to 12 months from the date of receipt. If you anticipate frequent use, prepare working aliquots (e.g., 5–10 µg per tube) at the time of first thaw. Purity is >95% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL assay, so degradation rather than endotoxin contamination is the primary concern to monitor over time.

Can I use recombinant Serpin B-5 as a positive control for Western blot with the RP-SerpinB5 antibody?

Yes — this is one of the most straightforward use cases. Load 50–100 ng of REC-SerpinB5 per lane alongside your cell lysate samples. The matched antibody RP-SerpinB5 (see /anti-serpin-b5-rabbit-polyclonal-antibody) was raised and validated in the same laboratory against the same protein, so band identity at 45–48 kDa is unambiguous. This pairing is particularly useful when validating Maspin expression in tumour tissue lysates where endogenous signal may be low or absent. Because both reagents originate from the same production run and epitope mapping effort, the recombinant serves as a reliable molecular weight marker and signal calibration reference.

How much recombinant Serpin B-5 should I load as a Western blot positive control, and what antibody dilution works?

For a robust positive control signal with RP-SerpinB5, load 50–150 ng of REC-SerpinB5 per lane on a standard 10–12% SDS-PAGE gel. At 50 ng, the band is typically visible with enhanced chemiluminescence at antibody dilutions of 1:1,000–1:2,000. Increasing to 100–150 ng provides a stronger reference band without causing blooming that obscures nearby molecular weight markers. For lysate lanes run in parallel, a total protein load of 20–40 µg from Maspin-expressing lines (MCF-7, 22Rv1) generally produces a comparable signal, aiding side-by-side comparison. Titrate the antibody to your specific ECL substrate and exposure conditions.

Which isoform or processing form of Serpin B-5 does this recombinant represent — is it full-length Maspin?

REC-SerpinB5 represents the canonical full-length human Serpin B-5 isoform (375 amino acids, UniProt P36952, isoform 1). No signal peptide cleavage or pro-domain processing is annotated for Maspin, so the expressed and purified product corresponds to the predominant cytoplasmic and nuclear form detected in epithelial tissues. Alternative splice isoforms have been described in the literature but are not recapitulated by this construct. If your experimental model involves a specific truncation or post-translational variant, confirm isoform identity against your cell line's transcript data before using this recombinant as a standard.