CAPN11 Antibody

What is CAPN11 and what is its general function in biological systems?

CAPN11 (Calpain-11) belongs to the calpain superfamily of calcium-dependent cysteine proteases. These proteases consist of a variable large (80 kDa) subunit and an invariant small (30 kDa) subunit . The CAPN11 gene was initially identified in human testis and contains motifs for calcium binding and protease activity . Research indicates CAPN11 plays a significant role in calcium-dependent signal transduction events during meiosis and sperm functional processes . Unlike many other calpain family members, CAPN11 appears to be uniquely expressed in spermatogenic cells, particularly in mouse models .

The calculated molecular weight of CAPN11 is approximately 80-83 kDa . Specifically, immunoblotting studies have identified CAPN11 as an 83 kDa protein in mouse testes extracts and spermatozoa . When working with these antibodies, researchers should expect to observe a band at approximately 80-83 kDa in Western blot applications, which aligns with the predicted size of the full-length protein .

What are the optimal storage conditions for CAPN11 antibodies?

For maximum stability and performance, CAPN11 antibodies should be:

• Stored at -20°C for long-term storage

• Aliquoted to avoid repeated freeze/thaw cycles that may compromise antibody function

• Shipped at 4°C, but transferred to -20°C upon receipt

• Stable for approximately one year after shipment when properly stored

Most CAPN11 antibodies are formulated in phosphate-buffered saline (PBS without Mg²⁺ and Ca²⁺, pH 7.4) containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol for stability .

How should researchers optimize Western blot protocols for CAPN11 detection?

For optimal Western blot results with CAPN11 antibodies:

• Sample preparation: Use fresh tissues or cells with appropriate protease inhibitors to prevent degradation of the target protein.

• Dilution optimization: Begin with the manufacturer's recommended dilution range (typically 1:500-1:2000) and optimize based on signal-to-noise ratio.

• Blocking conditions: Use 5% non-fat dry milk or BSA in TBST for blocking membranes to reduce background.

• Validation controls: Include positive controls such as testis tissue extracts or specific cell lines like PC-3 cells which have demonstrated CAPN11 expression .

• Peptide competition: Consider performing a peptide competition assay to confirm specificity, as demonstrated in mouse studies where staining was greatly reduced when CAPN11 antiserum was pre-incubated with the immunizing peptide .

Multiple studies have successfully detected CAPN11 in cell lines including HepG2, Jurkat, and HuvEc cells , as well as in mouse testis and isolated spermatozoa .

What is known about CAPN11 expression patterns during spermatogenesis?

Research using quantitative RT-PCR and immunoblotting has established a specific temporal expression pattern for CAPN11 during mouse spermatogenesis:

• mRNA expression timeline:

  • CAPN11 mRNA begins accumulating between days 14-16 after birth

  • Peak expression occurs around day 20, corresponding with pachytene spermatocyte appearance

  • Expression shows a decline on days 22-24, followed by an increase on day 26

• Protein expression timeline:

  • CAPN11 protein is first clearly detected on day 18 after birth, coinciding with late pachytene spermatocytes

  • Protein levels plateau around day 24

• Cellular localization:

  • Immunofluorescence combined with confocal microscopy suggests CAPN11 localizes to the acrosome in mouse spermatozoa

This expression pattern suggests CAPN11 plays specific roles during meiosis and in mature sperm function, making it an interesting target for reproductive biology research .

Researchers should be aware of several potential limitations when working with CAPN11 antibodies:

• Non-specific binding: Some CAPN11 antibodies may exhibit non-specific cross-reactivity. For example, in one study, an upper band (above 83 kDa) was consistently observed but was determined to be non-specific as it was not eliminated by peptide competition . Similarly, a lower band observed in kidney extracts was not eliminated by peptide blocking, suggesting non-specific cross-reaction .

• Variable performance across applications: While most CAPN11 antibodies perform well in Western blotting, their utility in other applications like IHC and IF may be more variable and require extensive optimization .

• Background issues: When using CAPN11 antibodies for immunofluorescence, researchers should always include appropriate controls (secondary antibody alone and peptide competition controls) to distinguish specific from non-specific staining .

• Limited validation in diverse tissues: Most validation has focused on reproductive tissues, with less information available about antibody performance in other contexts where CAPN11 might be expressed .

How can CAPN11 antibodies be used to investigate potential roles in cancer research?

Recent studies have begun exploring calpain family members as potential biomarkers in cancer:

• Prognostic significance: While specific data for CAPN11 is limited, other calpain family members (CAPN1, 2, 5, 8, and 10) have demonstrated prognostic value in pancreatic cancer . These findings suggest potential for investigating CAPN11's role in reproductive system cancers.

• Correlation with clinical parameters: Research has shown that some calpains correlate with clinical parameters such as:

  • Sex-specific prognostic value (e.g., CAPN8 only in female patients)

  • Age-dependent associations (e.g., stronger correlations in younger patients)

  • Correlations with smoking status and diabetes history

• Immune infiltration: Certain calpains have been correlated with tumor-infiltrating immune cells , suggesting a potential research direction for CAPN11 antibodies in investigating tumor microenvironment interactions in testicular or other reproductive system cancers.

What are the optimal protocols for CAPN11 immunohistochemistry in tissue sections?

For successful immunohistochemical detection of CAPN11:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde or 10% neutral buffered formalin

  • Process and embed in paraffin following standard protocols

  • Section at 4-6 μm thickness

• Antigen retrieval:

  • Use TE buffer pH 9.0 for optimal epitope exposure (recommended for mouse testis tissue)

  • Alternatively, citrate buffer pH 6.0 may be used, but comparison testing is advised

• Antibody incubation:

  • Use dilutions between 1:50-1:500 as recommended by manufacturers

  • Incubate overnight at 4°C for optimal binding

  • Include negative controls (no primary antibody) and peptide competition controls

• Detection systems:

  • Use biotin-streptavidin-HRP or polymer-based detection systems

  • Develop with DAB or other appropriate chromogens

  • Counterstain with hematoxylin for nuclear visualization

How can subcellular localization of CAPN11 be effectively studied using immunofluorescence?

Based on research findings that CAPN11 localizes to the acrosome in mouse spermatozoa , optimized immunofluorescence protocols should include:

• Sample preparation:

  • For sperm cells: Fix in 4% paraformaldehyde, permeabilize with 0.1% Triton X-100

  • For cell lines: Culture cells on coverslips, fix with 4% paraformaldehyde, permeabilize with 0.2% Triton X-100

• Antibody dilution and incubation:

  • Use at 1:200-1:800 dilution as recommended

  • Incubate overnight at 4°C

  • Include peptide competition controls to verify specificity

• Co-localization studies:

  • Consider double-labeling with acrosomal markers (for sperm studies)

  • Use confocal microscopy for precise subcellular localization

• Controls:

  • Secondary antibody alone (to assess non-specific binding)

  • Peptide-blocked primary antibody (to confirm specificity)

How might CAPN11 antibodies be used to investigate potential roles beyond reproductive biology?

While CAPN11 is primarily associated with testis expression and spermatogenesis, future research directions might include:

• Expression profiling in cancer: Investigating potential aberrant expression in various cancer types, given that other calpain family members have shown prognostic value in specific cancers .

• Potential roles in calcium signaling: As a calcium-dependent protease, exploring CAPN11's function in calcium signaling pathways beyond reproductive contexts.

• Comparative studies across species: Using antibodies to examine evolutionary conservation and divergence of CAPN11 function across species, particularly given the predicted reactivity with various mammalian species (cow, dog, goat, horse, etc.) .

What modifications to current CAPN11 antibodies might enhance their research utility?

Several potential improvements could advance CAPN11 antibody research applications:

• Development of monoclonal antibodies: Most current CAPN11 antibodies are polyclonal . Development of monoclonal antibodies could provide more consistent results with lower batch-to-batch variation.

• Phospho-specific antibodies: Given the potential regulatory role of phosphorylation in calpain activity, developing phospho-specific CAPN11 antibodies could provide insights into its regulation.

• Antibodies targeting different epitopes: Creating antibodies against various domains of CAPN11 could help elucidate domain-specific functions and interactions.

• Fluorophore-conjugated antibodies: Direct conjugation to fluorophores could enhance utility in flow cytometry and live-cell imaging applications.

• Increased validation across applications: More extensive validation across diverse techniques and samples would strengthen confidence in experimental results.