Calpain S-1 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for Calpain S-1 (Recombinant) on SDS-PAGE or Western blot?

Recombinant CAPNS1 (UniProt P04632) runs at approximately 28 kDa on reducing SDS-PAGE, consistent with its 268-amino-acid sequence (calculated MW ~28.3 kDa). Due to post-translational modifications acquired during HEK293 expression, the apparent band may shift slightly to 29–30 kDa on Western blot. Purity is >95% by SDS-PAGE under our standard QC conditions. A single predominant band at this position confirms protein integrity; a faint higher-MW band occasionally reflects residual SDS-resistant dimers, which is not indicative of degradation.

Is this recombinant the full-length CAPNS1 small subunit or a truncated fragment — does it include the penta-EF-hand domain?

This product is full-length human CAPNS1 (residues 1–268, UniProt P04632), expressed in HEK293 cells to preserve mammalian folding. It includes both the N-terminal glycine-rich domain V and the full penta-EF-hand domain required for calcium-dependent membrane association and holoenzyme stability. This matters functionally: truncations that remove even one EF-hand loop can impair calcium coordination and destabilize the heterodimer. For reconstitution experiments, full-length CAPNS1 is required to obtain a protease-competent calpain-1 or calpain-2 complex with the corresponding large subunit.

How do I reconstitute active calpain heterodimer in vitro using recombinant Calpain S-1?

Mix recombinant CAPNS1 with recombinant CAPN1 or CAPN2 large subunit in a 1:1 to 1.2:1 molar ratio in assembly buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT, 1 mM EDTA) on ice for 30 minutes. Confirm complex formation by native PAGE or size-exclusion chromatography before proceeding to activity assays. Initiate proteolysis by supplementing with CaCl2 to 0.05–0.5 mM (calpain-1/µ-calpain) or 0.3–1 mM (calpain-2/m-calpain). Avoid adding calcium during assembly, as premature activation can cause autolysis of the large subunit prior to heterodimerization.

What substrate and assay buffer should I use to measure calpain activity after reconstituting with recombinant CAPNS1?

For fluorogenic activity assays, use Suc-LLVY-AMC (25–100 µM) or the calpain-specific substrate Z-Leu-Arg-AMC in assay buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT, and 0.5 mM CaCl2 for calpain-1 reconstituted complexes. Read fluorescence at Ex/Em 355/460 nm. A casein zymography assay (0.1% casein in native PAGE) is a useful orthogonal confirmation. Include 10 µM ALLN (calpain inhibitor I) as a negative control to confirm calcium-dependent, calpain-specific activity rather than contaminating protease activity.

What starting concentration of recombinant Calpain S-1 should I use in an inhibitor screening or IC50 assay?

For inhibitor screening, reconstitute the CAPNS1/CAPN1 heterodimer at 10–50 nM total complex in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT, 0.5 mM CaCl2). Pre-incubate the complex with inhibitor for 15–30 minutes at 25°C before initiating the reaction with substrate. Confirm that substrate concentration is well below Km to avoid competitive artifact. At 50 nM heterodimer with 50 µM Suc-LLVY-AMC, linear progress curves typically extend 20–40 minutes, providing a reliable window for IC50 determination. Titrate enzyme concentration empirically for each substrate lot.

Can I use Calpain S-1 (Recombinant) as a positive control for Western blot with TPB antibody RP-CalpainS1?

Yes — this is one of the primary intended uses. REC-CalpainS1 was produced in the same lab that raised anti-calpain S-1 rabbit polyclonal antibody RP-CalpainS1, and compatibility is guaranteed. Load 20–50 ng of REC-CalpainS1 per lane alongside your cell lysate samples, run under reducing SDS-PAGE, and expect a clean band at ~28–30 kDa with RP-CalpainS1 at a 1:500–1:2000 dilution. This protein control simultaneously validates antibody sensitivity and confirms band identity in your experimental lanes, particularly important when working with complex lysates where CAPNS1 signal may be obscured.

How much recombinant Calpain S-1 should I load for Western blot positive control, and will it interfere with my lysate lanes?

Load 20–50 ng of REC-CalpainS1 in a dedicated lane separate from your cell or tissue lysate lanes to avoid any interference. At 50 ng, the RP-CalpainS1 antibody (SKU: RP-CalpainS1) reliably detects a single band at ~28–30 kDa with minimal background on PVDF or nitrocellulose. If you are titrating antibody concentration, 20 ng is sufficient at the higher end (1:500). Avoid loading more than 100 ng, as band broadening at high protein load can complicate MW estimation relative to your endogenous CAPNS1 signal in adjacent lysate lanes.

How should I store Calpain S-1 (Recombinant) and what is its shelf life after thawing?

Store at –20°C in single-use aliquots in the supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol). Avoid repeated freeze-thaw cycles, which degrade EF-hand domain integrity and reduce calcium-binding efficiency. Once thawed, keep on ice and use within 4–8 hours; do not refreeze. Shelf life at –20°C is 12 months from date of receipt when stored correctly. If you require diluted working stocks (e.g., for assay plate setup), prepare them fresh in assay-compatible buffer with 0.1% BSA as carrier and use same-day. Do not store working dilutions overnight.