Anti-Calpain-15 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for Calpain-15 on Western blot?

Human Calpain-15 (CAPN15, UniProt O75808) runs at approximately 113 kDa on SDS-PAGE, corresponding to the full-length protein. Some researchers report additional bands around 95-100 kDa, likely representing proteolytic processing or alternative translation initiation. Because Calpain-15 itself is a protease, include protease inhibitors (EDTA, calpain inhibitors such as ALLN or calpeptin) in your lysis buffer to minimize autodegradation. Calcium-dependent activation during sample preparation can produce lower molecular weight fragments, so maintaining calcium-free conditions until deliberate activation is advisable.

What dilution should I start with for Western blot with this Calpain-15 antibody?

Start at 1:1000 dilution in blocking buffer (5% non-fat milk or BSA in TBST) for Western blot, based on our validation data with human lysates. This polyclonal antibody at 100 µg per vial provides sufficient material for approximately 100 blots at this working dilution. If signal is weak with low-abundance samples or non-human species, titrate to 1:500. Conversely, if background is high, dilute to 1:2000. Overnight incubation at 4°C typically yields better signal-to-noise than shorter room-temperature incubations for this target.

Does this antibody work in mouse and rat tissue samples?

Cross-reactivity with mouse and rat Calpain-15 is predicted but not experimentally validated by Triple Point Biologics. Human and mouse CAPN15 share approximately 85% sequence identity, and rat is similarly conserved, suggesting the antibody should recognize these orthologs. We recommend pilot experiments with known positive control tissues—Calpain-15 is expressed in developing brain, cerebellum, and retina in rodents. Include a human lysate positive control alongside your rodent samples for the first experiment. If no signal appears, titrate the antibody concentration before concluding it does not cross-react.

What positive control tissue or cell line should I use for Calpain-15?

Calpain-15 is enriched in tissues undergoing active development or remodeling, particularly fetal brain, retina, and cerebellum. For cell lines, consider those with high E-cadherin turnover or epithelial-mesenchymal characteristics, as Calpain-15 regulates E-cadherin proteolysis. Adult tissue expression is lower and more restricted than developmental stages. If your experimental system has low endogenous expression, consider transiently transfecting HEK293 or COS-7 cells with a CAPN15 expression construct as a robust positive control for antibody validation before proceeding to your samples of interest.

Can this antibody detect Calpain-15 in immunofluorescence or IHC?

Triple Point Biologics antibodies are validated for Western blot and suitable for IHC and immunofluorescence applications, though we have not generated specific dilution data for Calpain-15 in these formats. For IHC on paraffin sections, start with 1:100 to 1:200 dilution after heat-induced antigen retrieval in citrate buffer, pH 6.0. For immunofluorescence on fixed cells, try 1:200 to 1:500 in blocking buffer. Because Calpain-15 has both cytoplasmic and membrane-associated localization depending on activation state, permeabilization with 0.1-0.3% Triton X-100 is necessary for full detection.

How should I prepare cell lysates to preserve Calpain-15 for Western blot?

Use calcium-free lysis buffer with EDTA or EGTA (5-10 mM) to prevent calcium-dependent autocatalysis during extraction, since Calpain-15 is itself a calcium-activated protease. Include a broad-spectrum protease inhibitor cocktail plus specific calpain inhibitors (ALLN at 10-25 µM or calpeptin at 20 µM) if possible. Lyse on ice and proceed to denaturation quickly. Avoid freeze-thaw cycles of undenatured lysates, as this can activate residual protease activity. If studying calcium-dependent processing, prepare parallel samples with and without calcium/inhibitors to demonstrate specific proteolytic regulation by Calpain-15.

Does Calpain-15 require ubiquitinated substrates for detection, or can I detect the enzyme itself?

This antibody detects Calpain-15 protein itself, regardless of whether it is bound to ubiquitinated substrates. You are detecting the protease, not its activity or substrate complex. Total Calpain-15 levels can be assessed in standard lysates. However, if your experimental goal is to study substrate interaction, consider immunoprecipitation of Calpain-15 followed by blotting for ubiquitin or specific substrates like E-cadherin. Calpain-15 activity is calcium-dependent and enhanced by substrate ubiquitination, but neither is required for the antibody to bind its epitope on the Calpain-15 polypeptide during Western blot or IHC.

How should I store this antibody and what is the expected shelf life?

Store the antibody at -20°C upon receipt. The 100 µg format in glycerol-containing buffer is stable for at least 12 months at -20°C and tolerates multiple freeze-thaw cycles, though minimizing freeze-thaw to fewer than five cycles is advisable for optimal performance. For frequent use, aliquot into single-use volumes (10-20 µL) immediately after receipt and store at -20°C. Avoid prolonged exposure to room temperature. Do not store diluted antibody in working solution; prepare fresh dilutions for each experiment. If you observe decreased signal over time, titrate to a higher concentration before assuming the antibody has degraded.