Anti-Calpain-5 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Calpain-5 on Western blot?

Calpain-5 migrates at approximately 70 kDa on SDS-PAGE, corresponding to the full-length 640-amino acid protein. Some lysates may show additional bands around 55-60 kDa representing proteolytic fragments or alternative processing. Retinal tissue lysates typically yield the cleanest signal given CAPN5's high endogenous expression in this tissue. If working with transfected cell lines, expect a single band at 70 kDa. We recommend starting at 1:1000 dilution and adjusting based on your protein load and detection system sensitivity.

Is this antibody validated for mouse and dog samples or only human?

This antibody has been validated with human samples. Mouse and dog are listed as potential cross-reactive species based on epitope homology, but have not been experimentally validated in our hands. Human CAPN5 shares approximately 85% identity with mouse and 92% with dog orthologs. If working with these species, we recommend testing at the standard 1:1000 dilution alongside a human positive control. Retinal lysates provide the most reliable positive tissue for cross-reactivity assessment given CAPN5's restricted expression profile.

What tissue or cell line works best as a positive control for Calpain-5?

Human retinal tissue or retinal pigment epithelium preparations serve as the strongest positive controls due to CAPN5's high expression in retina. Testis lysate is an alternative, as CAPN5 shows enriched expression there as well. Most commonly used cell lines express little to no detectable Calpain-5, making them poor choices for validation. If retinal tissue is unavailable, consider transiently transfecting HEK293 or COS cells with a CAPN5 expression construct to generate a reliable positive control for your Western blots and immunofluorescence experiments.

Does Calpain-5 require calcium in the lysis buffer for proper detection?

No. Unlike classical calpains that bind calcium for activation, you do not need to supplement lysis buffers with calcium to detect CAPN5 by Western blot. Standard RIPA or NP-40 lysis buffers with protease inhibitors work well. In fact, including EDTA or EGTA in your lysis buffer is advisable to inhibit endogenous calpain activity that might degrade CAPN5 or other targets during sample preparation. The antibody recognizes the native folded epitope and denatured forms equally well under reducing SDS-PAGE conditions.

Can I use this antibody for immunohistochemistry on retinal sections?

Yes. Triple Point Biologics antibodies are validated for Western blot; IHC application data is in progress, and given CAPN5's high retinal expression, this antibody should perform well on human retinal cryosections or paraffin-embedded tissue. Start with a 1:200 to 1:500 dilution for IHC, as this typically requires higher antibody concentration than Western blot. Antigen retrieval may improve signal depending on your fixation protocol. Expect predominant staining in photoreceptor and retinal pigment epithelium layers. Include a no-primary-antibody control to assess background in your detection system.

How should I distinguish Calpain-5 from other calpain family members on a blot?

Calpain-5's 70 kDa size distinguishes it from calpain-1 and calpain-2, which migrate around 80 kDa as heterodimers with the common regulatory subunit. Calpain-10 runs at approximately 75 kDa, close to CAPN5. The most definitive approach combines molecular weight with tissue distribution: CAPN5 should be abundant in retina and testis but absent or very low in ubiquitously expressing tissues like liver, brain, or skeletal muscle where classical calpains dominate. Running retinal lysate alongside a negative tissue like liver provides clear specificity confirmation.

What are common reasons for weak or absent Calpain-5 signal in Western blot?

The most common issue is working with tissues or cell lines that genuinely lack CAPN5 expression. Verify you are using retina, testis, or validated positive control lysates. Second, ensure adequate protein loading: 20-50 µg total protein is a reasonable starting range for retinal lysates. Third, confirm your detection system sensitivity, particularly if using standard chemiluminescence rather than enhanced substrates. If signal remains weak with validated positive controls, try a more concentrated dilution such as 1:500. Avoid over-diluting lysates, as CAPN5 is tissue-restricted and not abundant even in positive samples.

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

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer over time. If you use the antibody frequently, a working aliquot can be kept at 4°C for up to one month with 0.02% sodium azide as preservative. Do not store diluted antibody in blocking buffer for more than one week. Under proper storage at -20°C, rabbit polyclonal antibodies typically maintain activity for 2-3 years from the manufacture date. Avoid exposure to room temperature for extended periods during experiments.