Calpain-10 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Calpain-10 on SDS-PAGE or Western blot?

Recombinant Calpain-10 (REC-Calpain10) is a 672-amino-acid protein with a predicted molecular weight of approximately 74–76 kDa. On reducing SDS-PAGE, the major band typically migrates between 75 and 80 kDa due to post-translational modifications supported by the HEK293 expression system. Purity is >95% by SDS-PAGE. When using this as a Western blot standard alongside our matched antibody RP-Calpain10, expect a clean, dominant band in that range with minimal degradation products if handled correctly.

Is this recombinant Calpain-10 the full-length protein or a processed/truncated isoform?

REC-Calpain10 is produced as the full-length 672-amino-acid human sequence (UniProt Q9HC96), expressed in HEK293 cells to support native-like folding and post-translational processing. Unlike classical calpain-1/2, Calpain-10 does not require heterodimerization with a regulatory small subunit (CAPNS1) for activity, so the full-length monomer represents the physiologically relevant catalytic form. No artificial truncations have been introduced; the protein retains all three calpain-type domains (I–III) plus the C-terminal domain III-like region.

What substrates does Calpain-10 cleave and which fluorogenic peptide substrate works for activity assays?

Calpain-10 is a calcium-activated cysteine protease with activity toward cytoskeletal and signaling proteins implicated in insulin-stimulated glucose uptake. For in vitro fluorogenic assays, the Suc-LLVY-AMC substrate (used broadly for calpains) provides a measurable fluorescent readout; however, because Calpain-10 has divergent substrate specificity relative to calpain-1/2, the Z-LR-AMC substrate is also reported to give a stronger signal-to-noise ratio. Reactions should be run at 25–37°C in the presence of 1–5 mM CaCl₂ in assay buffer, with enzyme at 50–200 nM as a starting range.

What buffer conditions should I use for a Calpain-10 activity assay and does the storage buffer interfere?

REC-Calpain10 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For activity assays, dilute into a working buffer of 20–50 mM Tris-HCl pH 7.4 or HEPES pH 7.4, 1–5 mM CaCl₂, 1–5 mM DTT or β-mercaptoethanol (to maintain the active-site cysteine in reduced form), and 0.1% BSA as a carrier. Glycerol carry-over at dilutions of 1:10 or greater is generally non-inhibitory. Avoid EDTA or EGTA, which chelate the calcium required for activation. Pre-incubate the enzyme with calcium for 5–10 minutes at room temperature before adding substrate.

What starting concentration of recombinant Calpain-10 should I use in a substrate cleavage or IC50 inhibitor assay?

For fluorogenic substrate cleavage assays (e.g., Suc-LLVY-AMC or Z-LR-AMC), a starting concentration of 50–200 nM REC-Calpain10 in a 50–100 µL reaction volume is recommended. Titrate down if background hydrolysis is high, or up if signal is weak after 30–60 minutes. For IC50 determinations, fix enzyme at a concentration giving 20–30% substrate conversion at the assay endpoint to maintain linear kinetics. Include a no-enzyme blank and a no-inhibitor control. Confirm protein concentration by absorbance (A₂₈₀) before each use, as freeze-thaw losses can occur even with single-use aliquots.

Can I use REC-Calpain10 as a positive control for Western blot with the RP-Calpain10 antibody?

Yes — REC-Calpain10 is the intended positive control for the matched rabbit polyclonal antibody RP-Calpain10 (/anti-calpain-10-rabbit-polyclonal-antibody). Because both products originate from the same laboratory workflow, antigen–antibody compatibility is confirmed, not inferred. Load 10–50 ng of recombinant protein per lane on a standard SDS-PAGE gel; the antibody should detect the ~75–80 kDa band cleanly at antibody dilutions of 1:500–1:2000. This pairing is particularly useful for antibody validation experiments and for confirming band identity in complex lysate backgrounds where an endogenous Calpain-10 band needs unambiguous assignment.

How much recombinant Calpain-10 should I load for a Western blot positive control lane?

Load 10–50 ng of REC-Calpain10 per lane as a starting range. At 10 ng with the RP-Calpain10 antibody at 1:1000, a strong single band at ~75–80 kDa is typically resolved without saturating the signal. If your lysate lanes are loaded at 20–40 µg total protein, 25 ng of recombinant is a practical midpoint that keeps the positive control band comparable in intensity to the endogenous signal. Running the recombinant alongside a molecular weight ladder and the antibody-validated lysate in the same blot provides a clear, publishable positive control lane.

How should I store and handle recombinant Calpain-10 to preserve activity over time?

REC-Calpain10 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and is aliquoted for single use. Store at -20°C; avoid repeated freeze-thaw cycles, which progressively reduce cysteine protease activity. Upon thawing, keep on ice and use within 4–6 hours. Do not dilute working stocks in plain water — use a carrier buffer containing 0.1% BSA and 1 mM DTT to minimize surface adsorption and oxidation of the active-site cysteine. If long-term stability data are needed for your specific assay conditions, a short activity time-course after initial thaw is advisable before committing large volumes to a screening run.