Calpain-1 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant Calpain-1 on SDS-PAGE or Western blot?

The full-length human Calpain-1 catalytic subunit (CAPN1, 714 aa) runs at approximately 82 kDa under denaturing, reducing SDS-PAGE conditions. Because this recombinant is expressed in HEK293 cells, glycosylation or other post-translational modifications may shift the apparent MW slightly above the predicted 81.7 kDa — a band in the 82–85 kDa range is normal. Autolytic processing, if it occurs, can produce a truncated ~76 kDa species; this is not a degradation artifact but a known feature of activated calpain.

Is this recombinant the full-length Calpain-1 large subunit or a processed / autolytic fragment?

The product is supplied as the full-length 714-amino-acid human CAPN1 catalytic large subunit (UniProt P07384), expressed in HEK293 cells without the regulatory small subunit CAPNS1. It does not arrive pre-complexed as the μ-calpain holoenzyme. Calcium-dependent autolysis at the N-terminal domain I is an intrinsic property of calpain-1; under assay conditions with ≥10 µM Ca²⁺, a minor autolytic fragment (~76 kDa) may appear alongside the intact 82–85 kDa band. For structural studies requiring the intact form, keep Ca²⁺ below activating concentrations during handling.

What substrates does recombinant Calpain-1 cleave and which fluorogenic substrate should I use for activity assays?

Calpain-1 is a cysteine protease (EC 3.4.22.52) with a preference for hydrophobic residues at P2. The most commonly used fluorogenic substrate is Suc-LLVY-AMC (also used for some proteasome assays — confirm with a calpain-specific inhibitor control such as PD150606 or ALLN). Alternatively, t-Boc-LM-CMAC and the cell-permeable substrate Boc-LM-7-amino-4-chloromethylcoumarin work well in vitro. Natural protein substrates include spectrin, talin, and focal adhesion kinase. For a clean kinetic readout, Suc-LLVY-AMC at 50–200 µM in activity buffer provides reliable linear-range fluorescence at Ex/Em 380/460 nm.

What buffer conditions should I use to measure Calpain-1 activity, and how much calcium is needed for activation?

A standard in vitro activity buffer for Calpain-1 is 50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, with free Ca²⁺ titrated using a CaCl₂/EGTA buffer system. Calpain-1 (μ-calpain) requires micromolar calcium — typically 5–50 µM free Ca²⁺ — for half-maximal activation. Note that the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is calcium-free to keep the enzyme quiescent; add calcium only at the time of the assay. DTT or TCEP (0.5–1 mM) is required to maintain the active-site cysteine (Cys115) in the reduced state.

What starting concentration of recombinant Calpain-1 should I use in a fluorogenic substrate activity assay?

For a 96-well plate format using Suc-LLVY-AMC (100 µM substrate), a starting enzyme concentration of 50–200 nM recombinant Calpain-1 in a 100 µL reaction volume typically gives a robust linear signal over 30–60 minutes at 37°C. Titrate down if the signal saturates early; titrate up if activity is below the linear range. Because activity depends on free Ca²⁺, run a Ca²⁺ concentration-response curve (0.1–100 µM) in your first experiment to establish EC₅₀ under your specific buffer conditions. Include a no-enzyme background and a calpastatin or ALLN inhibitor control in every plate.

Can I use recombinant Calpain-1 as a Western blot positive control with the matched TPB anti-Calpain-1 antibody (RP-Calpain1)?

Yes — this is a direct use case for SKU RP-Calpain1 (/anti-calpain-1-rabbit-polyclonal-antibody). The recombinant and the antibody are developed from the same CAPN1 sequence in the same lab, so epitope compatibility is confirmed, not merely predicted. Load 50–100 ng of recombinant Calpain-1 per lane alongside your cell or tissue lysate; a clean band at 82–85 kDa validates that RP-Calpain1 detects the correct molecular weight in your hands. This is particularly useful when running CAPN1 antibody validation experiments or troubleshooting faint endogenous signal in low-expression samples.

How much recombinant Calpain-1 should I load for a Western blot positive control lane?

50–100 ng per lane is sufficient for a strong, clean band when using RP-Calpain1 at its recommended working dilution (typically 1:500–1:2000; confirm on the antibody datasheet). Avoid loading more than 200 ng — excess recombinant can bleed into adjacent sample lanes and complicate quantification. Run the recombinant in a dedicated outer lane, separated from experimental samples by at least one empty lane if your gel format allows. The expected band is 82–85 kDa. If you see a secondary band at ~76 kDa, this is the known autolytic fragment, not a non-specific signal.

How should I store and handle recombinant Calpain-1 to preserve enzymatic activity, and what is its shelf life?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — store at -20°C in single-use aliquots. Repeated freeze-thaw cycles progressively reduce activity; plan aliquot volumes around single-experiment use. At -20°C in the supplied buffer, activity is stable for at least 12 months from the date of receipt. On ice during active use, the enzyme is stable for 4–6 hours. Do not dilute into calcium-containing buffers until immediately before the assay to prevent premature autolysis. If you notice loss of activity, verify the reducing agent (DTT or TCEP) in your working buffer is fresh.