Calpain-13 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Calpain-13 on SDS-PAGE and Western blot?

The full-length human Calpain-13 protein is 669 amino acids, predicting a theoretical MW of approximately 74 kDa. On reducing SDS-PAGE under our standard conditions, the recombinant band typically migrates between 75–80 kDa — slight upward shift from predicted MW is common for HEK293-expressed proteins due to glycosylation or other post-translational modifications. Purity is >90% by SDS-PAGE. When running a Western blot positive control alongside our matched rabbit polyclonal antibody RP-Calpain13, expect a clean primary band in this 75–80 kDa window.

Is recombinant Calpain-13 the full-length protein or a processed active fragment?

This preparation corresponds to full-length human Calpain-13 (residues 1–669, UniProt Q6MZZ7), expressed in HEK293 cells to support native-like folding and post-translational processing. CAPN13 belongs to the atypical calpain subgroup and lacks the calmodulin-like regulatory domain found in classical calpains (CAPN1/2). No truncation or pro-domain removal has been engineered into this construct. Because atypical calpains are less well characterized with respect to autolytic processing, we recommend verifying intact-form activity in your specific assay system before assuming full autocatalytic maturation.

What substrates are suitable for measuring Calpain-13 protease activity in vitro?

As a cysteine protease (EC 3.4.22.-), Calpain-13 is amenable to fluorogenic substrates routinely used for calpain family members, such as Suc-LLVY-AMC or Z-FR-AMC. However, because CAPN13 substrate specificity has not been as thoroughly mapped as CAPN1/2, we recommend profiling activity against a panel of cysteine-protease fluorogenic peptides and confirming cleavage is abolished by a cysteine protease inhibitor such as E-64 (positive inhibitor control, 10 µM). Start with 0.5–2 µg of recombinant Calpain-13 per 100 µL reaction and optimize from there.

What buffer conditions should I use for Calpain-13 activity assays, and does it require calcium?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — a standard cysteine protease-compatible buffer. Classical calpains require millimolar calcium for activation; CAPN13's calcium dependence has not been definitively established. For initial activity assays, test both calcium-supplemented (0.5–2 mM CaCl2) and calcium-free conditions. Include 1–5 mM DTT or β-mercaptoethanol to maintain the catalytic cysteine in reduced form. Avoid EDTA at concentrations above 1 mM in the assay well, as it may chelate any free calcium needed for partial activation.

What starting enzyme concentration should I use for IC50 determination with Calpain-13?

For inhibitor screening and IC50 calculations, start with 0.5–1 µg recombinant Calpain-13 per 100 µL reaction — this typically provides a signal-to-background ratio sufficient for robust curve fitting without saturating substrate turnover. Pre-incubate the enzyme with inhibitor for 15–30 minutes at room temperature before adding substrate. Confirm that substrate concentration is at or below the Km for your chosen peptide, and that DMSO (if used as inhibitor vehicle) does not exceed 0.5% v/v, as higher concentrations can affect cysteine protease activity.

Can I use recombinant Calpain-13 as a positive control for Western blot with the RP-Calpain13 antibody?

Yes — this is one of the primary intended uses of this preparation. The matched rabbit polyclonal antibody RP-Calpain13 (TPB catalog /anti-calpain-13-rabbit-polyclonal-antibody) was validated in the same lab using this recombinant as the reference antigen, making cross-reactivity assured on Western blot. Load 50–100 ng of recombinant Calpain-13 per lane alongside your lysate samples. You should see a clean band at ~75–80 kDa. This confirms antibody sensitivity and correct band identification before interpreting endogenous expression levels in your experimental samples.

How much recombinant Calpain-13 should I load for Western blot positive control, and what antibody dilution pairs with it?

Load 50–100 ng per lane for a strong, clean positive control signal. For lysate comparisons, 25–50 ng is generally sufficient since the recombinant is a near-homogeneous preparation versus total protein complexity in cell lysates. Pair with RP-Calpain13 at 1:500–1:2000 dilution in 5% BSA/TBST; the optimal dilution for your detection system (HRP vs. fluorescent secondary) should be titrated empirically. Because RP-Calpain13 was raised and validated against the same HEK293-expressed antigen, non-specific background in this lane should be minimal.

How should I store and handle recombinant Calpain-13 to preserve activity, and what is the shelf life?

Store at -20°C in single-use aliquots immediately upon receipt. The supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is formulated for long-term frozen storage; glycerol prevents freeze-induced aggregation. Avoid repeated freeze-thaw cycles — each cycle can reduce cysteine protease activity measurably. When thawing, place on ice and use within the same working session. Shelf life is 12 months from receipt at -20°C when stored properly. For dilutions into assay buffer, prepare fresh on the day of use and keep on ice; do not re-freeze diluted material.