Anti-Calpain-10 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Calpain-10 on Western blot?

Full-length human Calpain-10 (CAPN10, UniProt Q9HC96) runs at approximately 75 kDa, corresponding to the 672-amino acid polypeptide. You may observe additional bands representing splice variants or proteolytic fragments, as calpains can undergo autolysis. Some researchers report detection of processed forms in the 60-65 kDa range depending on cell type and activation state. If you see multiple bands, consider comparing samples prepared with and without protease inhibitors to distinguish genuine isoforms from degradation products. The expected dominant band is 75 kDa under reducing conditions.

Does this antibody work for mouse or rat Calpain-10 detection?

This antibody is validated for human Calpain-10. Cross-reactivity with mouse and rat orthologs is predicted based on sequence homology—human CAPN10 shares approximately 85-90 percent identity with rodent sequences—but we have not empirically validated detection in these species. If you are working with mouse or rat samples, we recommend titrating the antibody starting at 1:500 dilution and including a positive control human lysate on the same blot for comparison. Empirical testing in your system is necessary to confirm cross-species performance.

What is the recommended starting dilution for Western blot with this Calpain-10 antibody?

Start at 1:1000 dilution in blocking buffer (5 percent non-fat milk or BSA in TBST) for Western blot applications. This rabbit polyclonal typically provides clean signal at this dilution with overnight incubation at 4°C. If signal is weak, titrate to 1:500; if background is high, extend to 1:2000. Signal intensity will vary with Calpain-10 expression level—the protein is relatively low-abundance in many cell types. Lysates from pancreatic islets or skeletal muscle generally yield stronger signal than fibroblasts or HEK293 cells.

Which cell lines or tissues have high Calpain-10 expression for positive controls?

Calpain-10 is expressed broadly but at relatively low levels in most tissues. Skeletal muscle, pancreatic islets, liver, and kidney show moderate to high expression and serve as reliable positive controls. Among common cell lines, C2C12 myoblasts and differentiated myotubes express detectable levels. INS-1 or MIN6 pancreatic beta cells are also suitable. HeLa and HEK293 cells express lower levels and may require longer exposure times. For Western blot optimization, we recommend using human skeletal muscle lysate or differentiated C2C12 lysate as your initial positive control.

Can this antibody distinguish between active and inactive Calpain-10?

No, this antibody recognizes Calpain-10 regardless of activation state—it detects the protein by linear epitope, not conformational changes associated with calcium binding or catalytic activity. Calpain-10 activation involves calcium-dependent conformational shifts rather than cleavage-based activation like some other proteases. If you need to assess Calpain-10 activity rather than expression, consider pairing immunodetection with functional assays using fluorogenic calpain substrates or by monitoring proteolysis of known Calpain-10 substrates in your lysates. This antibody will report total protein abundance only.

What sample preparation is critical for detecting Calpain-10 without degradation?

Calpain-10 is a cysteine protease and can undergo autolysis if calcium levels rise during lysis. Prepare lysates in buffers containing EDTA or EGTA (5-10 mM) to chelate calcium and include a cysteine protease inhibitor cocktail or E-64 at minimum. Work quickly on ice and snap-freeze aliquots to avoid freeze-thaw cycles. Avoid calcium-containing lysis buffers. If you observe smearing or lower MW bands, this often reflects autolysis during sample handling. For IHC applications, standard formalin fixation and antigen retrieval (citrate buffer, pH 6.0) are appropriate.

How should I store this Calpain-10 antibody and what is the shelf life?

Store the antibody at -20°C in the supplied glycerol-containing buffer. Under these conditions, the antibody remains stable for at least 12 months from the date of receipt. Avoid repeated freeze-thaw cycles—if you use the antibody frequently, aliquot into single-use volumes upon arrival. For short-term use (up to one month), the antibody can be held at 4°C with 0.02-0.05 percent sodium azide as preservative. Do not freeze diluted working solutions. If you observe increased background or reduced signal intensity over time, this typically indicates antibody degradation and a fresh aliquot should be thawed.

Are there known Calpain-10 isoforms I should be aware of on Western blot?

Human CAPN10 has multiple splice variants documented in sequence databases, with isoform 1 (canonical 672 amino acids, 75 kDa) being the predominant form. Shorter isoforms arising from alternative splicing or proteolytic processing may appear as lower MW bands, though functional characterization of variants remains incomplete. The literature reports tissue-specific splicing, so band patterns may differ between skeletal muscle, pancreas, and other tissues. If you detect multiple bands, verify they are specific by peptide competition or siRNA knockdown. This polyclonal antibody likely recognizes epitopes shared across isoforms, so you may detect variants if they are expressed.