Calpain-6 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant Calpain-6 on SDS-PAGE or Western blot?

The full-length Calpain-6 construct (641 amino acids, UniProt Q9Y6Q1) has a predicted molecular weight of approximately 73 kDa. On reducing SDS-PAGE, the recombinant runs at roughly 75–78 kDa due to the C-terminal His-tag and typical SDS-binding behaviour of this protein. Purity is >95% by Coomassie-stained SDS-PAGE. When using this protein as a Western blot standard alongside our matched antibody RP-Calpain6, a single dominant band in the 75–80 kDa range is the expected outcome.

Does recombinant Calpain-6 have proteolytic activity and what substrates should I use to measure it?

Calpain-6 is a non-proteolytic calpain family member — the catalytic triad cysteine and flanking residues critical for cysteine-protease activity are not conserved in CAPN6, and no peptidase activity has been demonstrated in the literature. Standard calpain fluorogenic substrates such as Suc-LLVY-AMC are not appropriate here. If you are studying Calpain-6 function, the recommended assays are microtubule co-sedimentation (to assess stabilisation activity) or pull-down / co-IP assays interrogating interaction with ARHGEF2 or RAC1, rather than enzymatic cleavage assays.

What is the recommended buffer and storage condition for Calpain-6 (Recombinant) to maintain protein stability?

This recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. Store at −20°C in single-use aliquots to avoid activity loss from repeated freeze-thaw cycles. Note that unlike calpain metalloproteinases, this formulation does not include CaCl₂, consistent with Calpain-6's non-proteolytic nature. For assays requiring dilution into a working buffer, maintain pH 7.2–7.5 and include at least 0.1% BSA as a carrier to limit adsorption to tube walls at low protein concentrations.

How much recombinant Calpain-6 should I load as a positive control for Western blot?

For Western blot positive control alongside the matched antibody RP-Calpain6, 20–50 ng per lane is typically sufficient to generate a strong, clean band at approximately 75–78 kDa. If signal is too high, titrate down to 10 ng. Load alongside your cell or tissue lysates in adjacent lanes; this directly confirms antibody specificity and validates the expected molecular weight in your own gel system. Avoid loading more than 100 ng, as overloading can produce diffuse bands that complicate molecular weight estimation.

Can I use Calpain-6 (Recombinant) to validate the RP-Calpain6 rabbit polyclonal antibody by Western blot?

Yes — this is one of the primary use cases. The recombinant (REC-Calpain6) and the matched antibody (RP-Calpain6) are produced and validated in the same laboratory, so compatibility for Western blot is guaranteed. Run 20–50 ng of recombinant protein alongside your experimental lysates; RP-Calpain6 should detect the ~75–78 kDa band cleanly. This pairing is especially useful when establishing Calpain-6 expression in fetal or placental tissue lysates, where endogenous signal can be ambiguous without a confirmed positive control.

What starting concentration should I use for Calpain-6 (Recombinant) in microtubule co-sedimentation assays?

For microtubule co-sedimentation experiments assessing Calpain-6's stabilising activity, published protocols using non-proteolytic calpain family members typically start with 0.5–2 µM recombinant protein incubated with taxol-stabilised or dynamic microtubules in BRB80 buffer (80 mM PIPES pH 6.8, 1 mM MgCl₂, 1 mM EGTA). Titrate Calpain-6 across this range and quantify pellet-to-supernatant ratios by SDS-PAGE. Confirm that Calpain-6 is pelleting with microtubules — not due to aggregation — by including a no-microtubule control at each concentration point.

Which isoform or processing form of Calpain-6 does this recombinant represent?

This recombinant represents the canonical full-length isoform of human CAPN6 (UniProt Q9Y6Q1, 641 amino acids), produced by transient expression in HEK293 cells. HEK293 expression provides mammalian post-translational modifications including glycosylation patterns relevant to folding and solubility. No proteolytic auto-processing is expected, consistent with Calpain-6's non-proteolytic character. A single band on SDS-PAGE reflects the intact, unprocessed form. If your experimental model uses a specific splice variant or truncation, confirm isoform identity against the UniProt entry before ordering.

Can recombinant Calpain-6 be used in RAC1 GTPase or ARHGEF2 interaction pull-down assays?

Yes. Calpain-6 regulates RAC1 activity through interaction with the guanine nucleotide exchange factor ARHGEF2, making pull-down and co-IP assays a well-supported use case for this recombinant. The His-tagged recombinant can be immobilised on Ni-NTA resin for pull-down experiments with recombinant ARHGEF2 or RAC1, or used as bait in cell lysate pull-downs. Perform binding in physiological salt conditions (100–150 mM NaCl, pH 7.4) to preserve native interaction surfaces. Validate interacting bands by Western blot using RP-Calpain6 (SKU: RP-Calpain6) for the Calpain-6 component.