Serpin B-6 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Serpin B-6 on SDS-PAGE and Western blot?

The full-length 376-amino-acid sequence of Serpin B-6 (UniProt P35237) has a calculated molecular weight of approximately 42.6 kDa. On reducing SDS-PAGE, this HEK293-expressed recombinant typically resolves as a single band at ~44–46 kDa — the slight upward shift relative to theoretical MW is consistent with mammalian post-translational modifications. Purity is >90% by SDS-PAGE. When running Western blot positive controls alongside RP-SerpinB6 antibody validation, load this band as your reference at the 44–46 kDa position.

Does recombinant Serpin B-6 undergo any proteolytic processing or isoform variation I should account for?

Serpin B-6 is expressed as the canonical full-length 376-residue sequence without a signal peptide, consistent with its strictly cytoplasmic localisation. No known signal-peptide cleavage or pro-domain processing is expected. However, serpins can form a cleaved, relaxed conformation after stoichiometric proteinase engagement — so if your recombinant has been exposed to a target proteinase (e.g., during activity testing), you may observe a secondary band ~2–4 kDa lower on reducing SDS-PAGE, representing the cleaved inhibitory form. Fresh, proteinase-naive aliquots should show a single band.

Which proteinases does Serpin B-6 inhibit, and can I use it directly in an enzyme inhibition kinetic assay?

Serpin B-6 functions as a stoichiometric inhibitor of serine proteinases including cathepsin G, kallikrein-8, and thrombin that may access the cytosol through lysosomal leakage or membrane disruption. This recombinant is produced in active inhibitory form and is suitable for direct use in enzyme–inhibitor kinetic assays and IC50 determinations. Pair it with fluorogenic substrates appropriate to your target proteinase — for example, Suc-AAPF-AMC for cathepsin G, or tosyl-GPR-AMC for thrombin — monitoring substrate cleavage inhibition at 380/460 nm excitation/emission.

What buffer and assay conditions should I use for a Serpin B-6 inhibition kinetic assay?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — a buffer compatible with most serine proteinase activity assays without dilution-induced pH shifts. For kinetic experiments, dilute to working concentration in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20 or 0.1% BSA to prevent adsorption at low protein concentrations. Avoid calcium-free buffers if your target proteinase requires divalent cations. Glycerol at 10% should be tolerated by most fluorometric plate readers, but validate background fluorescence from carrier buffer using a no-protein control.

What starting concentration of recombinant Serpin B-6 should I use for IC50 determination against cathepsin G?

Because Serpin B-6 acts stoichiometrically rather than catalytically, IC50 experiments require molar-ratio titrations rather than classical Michaelis-Menten approaches. A practical starting point is a Serpin B-6 concentration range of 10–500 nM against a fixed cathepsin G concentration of 10–50 nM, with substrate (e.g., Suc-AAPF-AMC) at or below Km (~100 µM). Pre-incubate inhibitor and proteinase for 30 minutes at 37°C before adding substrate to allow complex formation. Adjust based on your enzyme lot's specific activity. The stoichiometry of inhibition (SI) should be characterised alongside apparent Ki for full kinetic characterisation.

Can I use Serpin B-6 (Recombinant) as a positive control for Western blot with the RP-SerpinB6 antibody?

Yes — this recombinant is the recommended positive control for RP-SerpinB6 (/anti-serpin-b6-rabbit-polyclonal-antibody). The antibody and recombinant are produced from the same lab pipeline, ensuring epitope compatibility is validated rather than assumed. Load 20–50 ng of recombinant per lane alongside your cell lysate samples. Under standard reducing conditions, expect a clean band at ~44–46 kDa. This pairing is particularly useful when validating RP-SerpinB6 in new cell or tissue lysate backgrounds where endogenous Serpin B-6 expression is uncertain or low.

How much recombinant Serpin B-6 should I load per lane for Western blot to avoid signal saturation with RP-SerpinB6?

For a standard ECL detection setup with RP-SerpinB6 at 1:1000 dilution, loading 20–50 ng of recombinant Serpin B-6 per lane typically yields a strong, non-saturated signal. If you are using high-sensitivity chemiluminescence substrates or HRP-conjugates with amplification, start at 5–10 ng to avoid blooming that obscures the band boundary. Run a two- to three-point titration (10, 25, 50 ng) alongside your lysate lanes on the first use to calibrate signal linearity and confirm the 44–46 kDa band position before committing to a fixed loading amount.

How should I store and handle recombinant Serpin B-6 to maintain inhibitory activity over time?

Store at -20°C in single-use aliquots as supplied. Repeated freeze-thaw cycles progressively denature serpins and can drive loop-sheet polymerisation, abolishing inhibitory activity without obvious change in SDS-PAGE band appearance. Upon first use, thaw on ice, briefly centrifuge (2,000 × g, 1 min) to collect any condensate, and dilute immediately into assay buffer. Do not leave protein at room temperature for more than 30 minutes before use. Working dilutions in BSA-supplemented buffer can be held at 4°C for up to 24 hours, but prepare fresh aliquots for each independent experiment for reproducible kinetic data.