Calpastatin (Recombinant)

Frequently Asked Questions

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

Full-length human Calpastatin (UniProt P20810, 708 aa) has a predicted molecular weight of ~76 kDa, but routinely migrates anomalously on SDS-PAGE at approximately 110–120 kDa due to its highly acidic, intrinsically disordered regions — a well-documented phenomenon for this protein. On reducing SDS-PAGE, our recombinant preparation (HEK293-expressed, >95% purity) resolves as a single band in that 110–120 kDa range. Do not assume underloading or degradation if the band runs high; this is intrinsic to Calpastatin's sequence composition, not an artifact of the expression system.

Does recombinant Calpastatin contain all four inhibitory domains, or is it a truncated fragment?

This recombinant corresponds to full-length human Calpastatin (residues 1–708), encompassing the N-terminal L-domain and all four homologous inhibitory repeat domains (domains I–IV). Each domain independently inhibits both µ-calpain (calpain-1) and m-calpain (calpain-2) in a calcium-dependent manner. Because the protein is expressed in HEK293 mammalian cells rather than E. coli, the conformational integrity relevant to multi-domain inhibitory activity is preserved. Researchers requiring sub-domain mapping or domain-specific IC₅₀ comparisons should note that all four inhibitory units are present and functional in this preparation.

What does recombinant Calpastatin inhibit, and how does it differ from a calpain substrate?

Calpastatin is an endogenous inhibitor, not a substrate — it does not get cleaved under standard assay conditions; it engages calpain in a calcium-dependent, non-competitive manner and blocks catalytic activity. It is highly specific to calpains (calpain-1 and calpain-2) and does not inhibit other cysteine proteases such as cathepsins or caspases at physiological concentrations. This specificity makes it the preferred tool for confirming calpain-dependent signal in activity assays: a band or fluorescence signal that is ablated by this recombinant but not by a broad cysteine protease inhibitor can be attributed specifically to calpain activity.

Which fluorogenic or chromogenic substrate works best for Calpastatin IC50 assays with calpain-1?

For fluorogenic calpain activity assays, Suc-LLVY-AMC (excitation ~380 nm, emission ~460 nm) at 50–200 µM in a calcium-containing buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM CaCl₂, 5 mM DTT) is the most widely used substrate. Casein-based substrates (e.g., BODIPY-casein or radiolabeled casein) are an alternative for gel-shift or fluorescence dequenching formats. When titrating recombinant Calpastatin against purified calpain-1, use a fixed enzyme concentration at or below its Km for the substrate to keep competitive effects minimal and derive a clean inhibition curve.

What starting concentration of recombinant Calpastatin should I use to inhibit calpain-1 in a fluorogenic assay?

A practical starting point is a 1:1 molar ratio of Calpastatin to calpain-1, typically 50–200 nM recombinant Calpastatin when using 50–100 nM purified calpain-1. Because Calpastatin carries four inhibitory domains, sub-stoichiometric concentrations (down to ~10–20 nM) may achieve substantial inhibition — titrate accordingly. Pre-incubate Calpastatin with calpain for 5–10 minutes at room temperature in the presence of 5 mM CaCl₂ before adding substrate, as calcium is required for Calpastatin–calpain complex formation. Omitting calcium will sharply reduce observed inhibition and confound your IC₅₀ estimate.

What buffer should I use to dilute recombinant Calpastatin for activity assays, and is the storage buffer compatible?

Recombinant Calpastatin is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For fluorogenic calpain activity assays, dilute into your assay buffer (e.g., 50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM DTT) to keep glycerol at ≤1% final concentration; glycerol above ~2% can modestly reduce AMC fluorescence read-out linearity. The Tris carry-over at typical working dilutions (≥1:10) is negligible. Note that CaCl₂ is absent from Calpastatin's storage buffer by design — add calcium in the assay itself to control the point of inhibitory complex formation.

How much recombinant Calpastatin should I load as a positive control for Western blot with the matched RP-Calpastatin antibody?

For a positive control lane alongside the matched rabbit polyclonal antibody RP-Calpastatin (/anti-calpastatin-rabbit-polyclonal-antibody), 20–50 ng of recombinant Calpastatin per lane is sufficient to produce a strong, clean band at ~110–120 kDa under standard reducing SDS-PAGE conditions. At 100 ng the band saturates most ECL exposures and can bleed into adjacent lanes. Run the recombinant alongside your cell or tissue lysates so the anomalous migration (~110–120 kDa vs. predicted 76 kDa) serves as an explicit molecular-weight reference for identifying endogenous Calpastatin in your sample.

Can I use recombinant Calpastatin to validate the RP-Calpastatin antibody before running my tissue lysates?

Yes — this is the most straightforward use case. Load a two-point dilution series (e.g., 25 ng and 100 ng) of recombinant Calpastatin alongside your lysate. If the RP-Calpastatin antibody detects a band co-migrating with the recombinant (~110–120 kDa) and signal scales linearly with load, antibody performance in your specific lysis/blot conditions is confirmed before you commit your primary tissue material. Because both the recombinant and the antibody are produced in the same TPB pipeline, lot-to-lot compatibility is verified internally — making this pairing reliable for antibody validation documentation.

How should I store recombinant Calpastatin and how many freeze-thaw cycles can it tolerate?

Store recombinant Calpastatin at −20°C in single-use aliquots. The supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) stabilizes the protein during freeze-thaw, but repeated cycling progressively compromises inhibitory activity. Aliquot on receipt to avoid re-freezing the master stock. Once thawed, working aliquots are stable for up to 48 hours at 4°C; do not leave at room temperature for extended periods. Shelf life from date of manufacture is 12 months at −20°C. Activity loss is not always visible by SDS-PAGE, so for IC₅₀-sensitive experiments, use fresh aliquots and confirm activity against a calpain standard each session.