Anti-Calpastatin Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for calpastatin on Western blot?

Full-length human calpastatin (CAST, UniProt P20810) runs at approximately 85 kDa, corresponding to the 708-amino acid polypeptide. You may observe additional bands between 60-75 kDa representing naturally occurring proteolytic fragments or alternative translation products. Calpastatin is subject to autolytic cleavage and calpain-mediated degradation in lysates if calcium is present, so include EDTA or EGTA in your lysis buffer. In postmortem or apoptotic samples, expect a ladder of lower-MW fragments. The four tandem inhibitory domains share sequence homology, so polyclonal antibodies may recognize multiple degradation products.

What dilution should I start with for Western blot with this calpastatin antibody?

We recommend starting at 1:1000 dilution for Western blot, based on validation with human skeletal muscle and heart lysates. Calpastatin expression varies widely by tissue—it is abundant in muscle (both skeletal and cardiac) but lower in many cultured cell lines. If working with HeLa, HEK293, or other non-muscle cells, you may need to concentrate your lysate or use 1:500 dilution. Load 20-30 µg total protein per lane as a starting point. For IHC applications, begin titration at 1:200 to 1:500 depending on fixation method and tissue type.

Is this antibody cross-reactive with mouse and dog calpastatin?

The antibody is validated for human calpastatin. Mouse and dog cross-reactivity are predicted based on sequence homology but not experimentally confirmed in our hands. Human and mouse calpastatin share approximately 88% identity across the full-length protein, with highest conservation in the inhibitory domains. If working with mouse or dog samples, treat this as a pilot experiment and include a positive control lysate from skeletal muscle, where calpastatin is most abundant. We recommend validating band identity by competition with blocking peptide or siRNA knockdown if species cross-reactivity is critical to your study.

What positive and negative control tissues should I use for calpastatin?

Skeletal muscle and cardiac muscle serve as excellent positive controls due to high endogenous calpastatin expression—these tissues rely on calpastatin to regulate myofibrillar protein turnover. Human or rodent heart lysate typically gives robust signal. For a negative control, consider calpastatin-null cells if available, though true negatives are rare since most mammalian cells express basal calpastatin. Alternatively, pre-incubate the antibody with excess blocking peptide (if available) to demonstrate specificity. Avoid using calcium-containing buffers during lysis, as they activate calpains that will degrade calpastatin and complicate interpretation.

Why do I see multiple bands for calpastatin on my Western blot?

Multiple bands are common with calpastatin and usually reflect proteolytic processing rather than cross-reactivity. The full-length 85 kDa protein is cleaved by calpains and other proteases into stable fragments retaining inhibitory activity. The four inhibitory domains (each ~17-20 kDa) can appear as discrete bands if extensive degradation occurs. To minimize artifactual proteolysis, lyse cells quickly in ice-cold buffer containing protease inhibitors (including calpain inhibitors like leupeptin or E64) and chelators (5 mM EDTA or EGTA). If bands persist under stringent conditions, they likely represent physiologically relevant isoforms or processing intermediates present in vivo.

Can I use this antibody for immunofluorescence of calpastatin?

Yes, this rabbit polyclonal is suitable for immunofluorescence, though optimization will be required. Calpastatin lacks a defined subcellular localization signal and distributes primarily in the cytosol, though it can translocate to membranes or associate with the cytoskeleton when binding active calpain. Use methanol or paraformaldehyde fixation and standard permeabilization (0.1-0.5% Triton X-100). Start with 1:100 to 1:200 dilution and expect diffuse cytoplasmic staining under basal conditions. In cells treated with calcium ionophore or undergoing apoptosis, you may observe redistribution or punctate staining reflecting calpain-calpastatin complexes at sites of active proteolysis.

Does calpastatin have multiple isoforms I should know about?

The human CAST gene produces several splice variants, though the full-length 708-amino acid Type I isoform is most widely studied. Type II and Type III variants have alternative N-termini and differ in the presence or absence of a nuclear localization signal encoded by exon 1. The four inhibitory domains (domains 1-4) are conserved across isoforms and confer redundant calpain-binding activity. Most polyclonal antibodies raised against the central region recognize all major isoforms. Functional differences between isoforms relate primarily to tissue distribution and subcellular targeting rather than inhibitory potency. If isoform-specific detection is required, epitope mapping relative to exon structure is necessary.

How should I store this calpastatin antibody and how long is it stable?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100 µg format typically arrives lyophilized or in glycerol-containing buffer—check the datasheet for your lot. Once reconstituted or thawed, the antibody remains stable for at least 6 months at -20°C and up to one year if stored properly. For day-to-day use, keep a working aliquot at 4°C for up to two weeks. Avoid prolonged storage in frost-free freezers due to temperature cycling. Add sodium azide (0.02-0.05%) as preservative for 4°C storage if not using for live-cell applications.