Calpain-8 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant Calpain-8 on SDS-PAGE or Western blot?

Recombinant Calpain-8 (REC-Calpain8) is expressed from the full 703-amino-acid open reading frame (UniProt A6NHC0), giving a predicted molecular weight of approximately 80 kDa. On reducing SDS-PAGE, the predominant band typically resolves between 78–85 kDa, consistent with HEK293-derived glycosylation and any minor post-translational modifications. No significant processing or autoproteolytic cleavage product is expected under standard denaturing conditions. Purity is confirmed at >90% by Coomassie-stained SDS-PAGE prior to release.

Is recombinant Calpain-8 the full-length protein or a catalytic domain fragment?

REC-Calpain8 is expressed as full-length human Calpain-8 (residues 1–703), not a truncated catalytic domain fragment. This distinction matters for calcium-binding geometry: the full-length construct retains the C2-domain-like region (domain III) and the penta-EF-hand domain (domain IV), both of which contribute to calcium-dependent conformational activation. Because HEK293 cells support the folding environment required for these domains, the recombinant is supplied as an active enzyme rather than a denatured or refolded construct.

What is the documented substrate for Calpain-8 and can I use it in a substrate cleavage assay?

The best-characterised substrate of Calpain-8 is the β-subunit of the COPI coatomer complex, implicating it in vesicular coat remodelling in gastric pit cells. For in vitro activity assays, fluorogenic peptide substrates commonly used across the calpain family — such as Suc-LLVY-AMC or Z-Leu-Arg-AMC — serve as surrogate substrates for initial activity verification and small-molecule screening. Cleavage is calcium-dependent; confirm activity with a no-calcium control. For direct substrate identification work, the recombinant's confirmed activity under native-like conditions makes it suitable for proteomic substrate-trapping approaches.

What buffer conditions and calcium concentration should I use for a Calpain-8 activity assay?

The storage buffer for REC-Calpain8 is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — formulated without added calcium to preserve enzyme stability during storage. For activity assays, dilute into a reaction buffer of 50 mM Tris-HCl pH 7.5, 50–100 mM NaCl, 1–5 mM CaCl₂, and 5–10 mM DTT (to maintain the active-site Cys in reduced form). Calpain-8 shows calcium-dependent activation; titrating CaCl₂ from 0.5–5 mM allows determination of half-maximal activation for your specific assay format. Keep DTT and calcium added fresh immediately before the reaction.

What starting concentration of recombinant Calpain-8 should I use in an enzyme inhibitor IC50 assay?

A practical starting point for IC50 determinations is 50–200 nM REC-Calpain8 in the reaction well, using a fluorogenic peptide substrate at 50–200 µM (well below or near its apparent Km to remain in a linear velocity regime). At this enzyme concentration, signal-to-background ratios with AMC-releasing substrates are typically adequate for 30–60 minute kinetic reads on a standard plate reader. Confirm linearity of product accumulation over time before committing to a full inhibitor titration. Adjust enzyme concentration downward if substrate depletion exceeds 10–15% of total during the read window.

Can I use recombinant Calpain-8 as a positive control for Western blot with the matched anti-Calpain-8 antibody?

Yes — this is one of the primary intended uses of REC-Calpain8 alongside the matched rabbit polyclonal antibody RP-Calpain8 (/anti-calpain-8-rabbit-polyclonal-antibody). Both were developed and validated in the same laboratory, ensuring epitope accessibility to the denatured recombinant on a membrane. Load 5–20 ng of REC-Calpain8 per lane alongside your lysate samples; a clean band at ~80 kDa confirms antibody performance and transfer efficiency. This pairing is particularly useful when working with non-gastric cell lines where endogenous Calpain-8 expression is low or absent.

How much recombinant Calpain-8 should I load per lane as a Western blot positive control?

For chemiluminescent detection with RP-Calpain8 at a 1:1,000–1:2,000 dilution, 5–15 ng of REC-Calpain8 per lane is sufficient to produce a well-resolved band at approximately 80 kDa without overwhelming adjacent lysate lanes. If using fluorescent secondary antibodies or a lower-sensitivity detection system, scale to 20–50 ng. Run the recombinant in a dedicated lane at the gel edge so it serves as both a size reference and a signal-strength benchmark. Avoid loading more than 50 ng, which can produce trailing artefacts that obscure migration relative to marker.

How should I handle, dilute, and store recombinant Calpain-8 to maintain activity over time?

REC-Calpain8 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol in single-use aliquots. Store at -20°C; avoid repeated freeze-thaw cycles, which progressively reduce cysteine-protease activity. For working dilutions, use the storage buffer itself or a compatible assay buffer — avoid diluting into pure water, which disrupts ionic strength and risks precipitation. Working solutions prepared on ice remain active for 4–6 hours. For experiments requiring dilutions below ~10 nM, add 0.1% BSA (protease-free) to the diluent to reduce adsorptive losses to tube surfaces.