Anti-Calpain-2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What band size should I expect for Calpain-2 on Western blot?

The Calpain-2 catalytic subunit (CAPN2) runs at approximately 80 kDa on reducing SDS-PAGE, corresponding to the 700-amino acid polypeptide. In some lysates you may observe additional bands around 76-78 kDa representing autolytic fragments, particularly if samples were not processed immediately or lack protease inhibitors. The antibody detects the full-length catalytic subunit; it does not recognize the separate 28 kDa regulatory subunit (CAPNS1) with which CAPN2 heterodimerizes in vivo. If you see lower molecular weight bands, consider whether calpain autoactivation occurred during sample preparation.

How do I distinguish Calpain-2 from Calpain-1 by Western blot?

Calpain-1 (CAPN1) and Calpain-2 (CAPN2) both run near 80 kDa and share approximately 60% sequence identity, so cross-reactivity is a legitimate concern. This antibody was raised against human CAPN2 and has been validated specifically against Calpain-2. To confirm specificity in your system, run lysates with known differential expression—skeletal muscle expresses relatively more Calpain-1, while some epithelial lines favor Calpain-2. Alternatively, use siRNA or CRISPR knockdown targeting CAPN2 specifically; the band should disappear or significantly diminish. We have not observed cross-reactivity with Calpain-1 in our validation panels.

What is the recommended starting dilution for Western blot with this Calpain-2 antibody?

Start at 1:1000 dilution in blocking buffer (5% non-fat milk or 3% BSA in TBST) for Western blot. This dilution has been validated with 20-30 µg total protein per lane from whole-cell lysates. If your signal is weak, you can increase antibody concentration to 1:500, but check for non-specific bands before committing to multiple blots. Conversely, if background is high or you are working with tissues that abundantly express Calpain-2, try 1:2000. Incubate overnight at 4°C or two hours at room temperature. The 100 µg pack provides sufficient antibody for approximately 100 blots at 1:1000.

Does this antibody work in mouse and rat samples?

The antibody is validated for human CAPN2 and predicted to react with mouse, rat, and dog based on sequence homology. Human and mouse CAPN2 share approximately 94% identity across the immunogenic region, and rat is similarly conserved. We have observed specific bands at the expected molecular weight in preliminary experiments with mouse brain lysate and rat kidney lysate, but formal validation in these species is ongoing. If you are working with rodent samples, we recommend including a positive control (human lysate) on the same blot and verifying the band disappears with a blocking peptide or CAPN2 knockdown in your model system.

What sample preparation considerations are critical for detecting Calpain-2?

Calpain-2 is itself a protease, so it can undergo autolysis if calcium levels rise during lysis. Lyse cells or tissues quickly in the presence of EDTA or EGTA (5-10 mM) to chelate calcium and prevent autoactivation. Include a broad-spectrum protease inhibitor cocktail, but avoid E-64 or leupeptin at high concentrations if you are also studying calpain activity in parallel experiments. Process samples on ice and snap-freeze aliquots immediately. Avoid multiple freeze-thaw cycles, which can promote aggregation and degradation. For tissues with high endogenous protease activity, consider snap-freezing in liquid nitrogen before homogenization.

Can I use this Calpain-2 antibody for immunofluorescence or immunohistochemistry?

Triple Point Biologics antibodies in this product line are validated for Western blot and immunohistochemistry as standard applications. This polyclonal antibody is suitable for IHC on formalin-fixed paraffin-embedded sections; antigen retrieval in citrate buffer (pH 6.0, 20 minutes sub-boiling) typically yields optimal results. For immunofluorescence, the antibody has been used successfully at 1:100 to 1:200 dilution, though we recommend titrating in your specific cell line and fixation protocol (4% paraformaldehyde, 10-15 minutes is a reasonable starting point). Calpain-2 localizes primarily to the cytoplasm under basal conditions but may translocate upon calcium influx or activation.

What positive and negative controls should I include when using this antibody?

HEK293 and HeLa whole-cell lysates serve as reliable positive controls; both express detectable endogenous Calpain-2. Brain and skeletal muscle tissue lysates also show robust expression. For a negative control, consider lysate from cells in which CAPN2 has been knocked down by siRNA (48-72 hours post-transfection) or knocked out by genome editing. Alternatively, pre-incubate the antibody with a blocking peptide corresponding to the immunogen (if available from the vendor) to demonstrate specificity. Including a no-primary-antibody control helps identify non-specific binding by the secondary antibody, particularly when optimizing IHC or IF protocols.

How should I store the Calpain-2 antibody and how long is it stable?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The antibody is supplied in a glycerol-containing buffer that remains liquid at -20°C, allowing you to remove small volumes without thawing the entire stock. If you prefer working stocks, prepare 50-100 µL aliquots and keep one at 4°C for up to one month; sodium azide (0.02% final) can extend this to three months but is incompatible with HRP-based detection. Under proper storage at -20°C, polyclonal antibodies typically retain activity for at least two years from the manufacture date.