CATSPER2 Antibody

What is CATSPER2 and what is its biological function?

CATSPER2 is one of four proteins that form a sperm-specific cation channel located principally on the sperm flagellum. It functions as a regulator of sperm motility by forming part of a pore that allows for the movement of charged calcium atoms (Ca²⁺) across the sperm cell membrane. This calcium influx is essential for hyperactivated motility, characterized by vigorous movements of the sperm tail that enable penetration through the egg's extracellular matrix during fertilization . Studies with CATSPER2-null mice have demonstrated that this protein is specifically required for the generation of hyperactivated motility, and that even with normal forward velocity and percentage of motile cells, fertilization fails without this hyperactivated form of movement .

How is CATSPER2 structured and where is it expressed?

CATSPER2 is a putative six-transmembrane voltage-gated ion channel. In humans, the canonical protein has a reported length of 530 amino acid residues and a mass of 62 kDa . Its subcellular localization is in the cell projections, specifically in the sperm flagellum. The protein exhibits testis-specific expression, with up to four different isoforms reported . CATSPER2 is a member of the Cation channel sperm-associated (TC 1.A.1.19) protein family and is expressed during late spermatogenesis .

What pathologies are associated with CATSPER2 dysfunction?

CATSPER2 gene disruption has been associated with deafness-infertility syndrome, a condition characterized by hearing loss and male infertility . In knockout mouse models, CATSPER2-null males are completely infertile due to the inability of sperm to generate hyperactivated motility . Recent research has also identified homozygous CATSPER2 deletions in twin brothers with unexplained infertility and hearing loss, affecting acrosome reaction and calcium mobilization during fertilization .

What types of CATSPER2 antibodies are available for research?

Various CATSPER2 antibodies are available targeting different regions of the protein, including:

• N-terminal region antibodies (e.g., AA 5-112)

• C-terminal region antibodies

• Mid-region antibodies (e.g., AA 31-80)

These antibodies come in different forms including:

• Unconjugated primary antibodies

• Conjugated antibodies (HRP, FITC, APC)

• Polyclonal and monoclonal varieties

• Species-specific antibodies with various reactivity profiles (primarily human, but some with cross-reactivity to mouse, rat, bovine and other species)

What are the optimal applications for CATSPER2 antibodies?

CATSPER2 antibodies have been validated for multiple applications with specific recommended dilutions:

Research indicates that Western Blot is the most commonly employed technique for CATSPER2 detection, allowing for confirmation of the predicted molecular weight (approximately 62 kDa) .

How should I optimize Western blot protocols for CATSPER2 detection?

When performing Western blots for CATSPER2 detection, consider these research-based recommendations:

• Sample preparation: Since CATSPER2 is a membrane protein, use detergent-based lysis buffers (such as those used in published CATSPER studies ).

• Protein loading: Load 20-50 μg of total protein from testicular tissue or sperm samples.

• Gel selection: Use 8-10% SDS-PAGE gels for optimal separation of the 62 kDa CATSPER2 protein.

• Transfer conditions: Employ wet transfer systems with methanol-containing buffers for efficient transfer of membrane proteins.

• Blocking: Use 5% non-fat milk or BSA (especially important for phospho-specific studies, as shown in CATSPER2 research examining tyrosine phosphorylation during capacitation ).

• Primary antibody incubation: Dilute antibodies in the range of 1:500-1:2000, and incubate overnight at 4°C for optimal results.

• Controls: Include appropriate positive controls (testicular tissue) and negative controls (tissues known not to express CATSPER2).

Research has shown that these conditions provide optimal signal-to-noise ratio for CATSPER2 detection in Western blot applications .

What are the critical considerations for immunolocalization of CATSPER2?

For accurate immunolocalization of CATSPER2 in sperm or testicular tissue:

• Fixation method: Use 4% paraformaldehyde for optimal preservation of membrane proteins while maintaining antigenicity.

• Permeabilization: Since CATSPER2 is a membrane protein, gentle permeabilization with 0.1-0.2% Triton X-100 is recommended.

• Blocking: Extended blocking (1-2 hours) with 5% BSA to minimize background.

• Antibody selection: Choose antibodies targeting extracellular domains for non-permeabilized samples, or intracellular domains for permeabilized samples.

• Co-localization studies: Consider dual labeling with flagellar markers to confirm specificity of localization.

• Microscopy technique: Use confocal microscopy for precise localization within the flagellar structure.

Research has demonstrated differential expression patterns of CATSPER2 along the flagellum, which is important for understanding its functional significance .

How can CATSPER2 antibodies be used to study male infertility?

CATSPER2 antibodies provide valuable tools for investigating male infertility through several methodological approaches:

• Diagnostic applications: Immunodetection of CATSPER2 in sperm samples from infertile men can identify potential deficiencies in this critical protein.

• Functional studies: Combining CATSPER2 immunodetection with calcium imaging can reveal correlations between CATSPER2 expression levels and functional calcium signaling.

• Pharmacological research: Antibodies can help assess how various compounds affect CATSPER2 expression and localization, potentially leading to fertility treatments.

• Genetic correlation studies: Linking genetic variants with protein expression patterns using antibody-based detection methods.

Research has established that CATSPER2 mutations are associated with male infertility, making antibody detection a valuable research tool for investigating the molecular basis of unexplained infertility cases .

What methodological approaches can be used to study the effects of environmental toxins on CATSPER2 expression?

Several experimental designs have been validated for studying environmental impacts on CATSPER2:

• Exposure models: Animal models (such as NMRI mice) can be exposed to environmental toxins (e.g., dioxin) at various doses (0.1-1 μg/kg) to assess effects on CATSPER2 expression .

• Expression analysis: Combine qRT-PCR for transcript levels with Western blot analysis using anti-CATSPER2 antibodies to correlate mRNA and protein changes.

• Histopathological correlation: Integrate antibody-based CATSPER2 protein detection with histopathological assessment of testicular tissue to correlate expression changes with tissue damage.

• Functional parameters: Correlate CATSPER2 expression levels with sperm parameters (motility, count) and oxidative stress markers.

Research has demonstrated that toxins like dioxin can significantly downregulate CATSPER2 gene and protein expression in a dose-dependent manner, with corresponding reductions in sperm motility and count .

How can I validate the specificity of CATSPER2 antibodies in my experiments?

To ensure specificity of CATSPER2 antibody detection:

• Positive controls: Use testicular tissue samples known to express CATSPER2.

• Negative controls: Include samples from tissues that don't express CATSPER2.

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to confirm specificity.

• Knockout/knockdown validation: When available, use samples from CATSPER2 knockout mice as definitive negative controls.

• Multiple antibody approach: Use antibodies targeting different epitopes of CATSPER2 to confirm findings.

• Recombinant protein controls: Include purified recombinant CATSPER2 protein as a positive control.

Research has employed careful validation of antibody specificity in CATSPER2 studies, particularly when studying knockout models .

What are common technical challenges when working with CATSPER2 antibodies and how can they be addressed?

Researchers commonly encounter these challenges when working with CATSPER2 antibodies:

• Background signal: Optimize blocking conditions (5% BSA or IgG-free BSA) and secondary antibody dilutions. Research has shown this is particularly important when examining phosphorylation states during capacitation .

• Low signal strength: Consider protein enrichment methods (membrane fractionation) to concentrate the target protein. Extend primary antibody incubation time (overnight at 4°C).

• Multiple bands: Verify if these represent known isoforms (up to 4 different isoforms have been reported ) or non-specific binding.

• Cross-reactivity: Choose antibodies with validated species-specificity for your research model. Some antibodies show cross-reactivity with multiple species including human, mouse, rat, bovine, and others .

• Limited tissue availability: When working with human samples, optimize protein extraction methods for maximal yield from limited material.

Research protocols typically incorporate these considerations when designing CATSPER2 antibody-based experiments .

How does CATSPER2 interact with other CATSPER family members?

This represents an active area of investigation with some contradictory findings:

• Complex formation: Although CATSPER1-4 are hypothesized to form a heterotetramer, attempts to co-immunoprecipitate CATSPER1 and CATSPER2 from detergent extracts have failed .

• Expression patterns: Immunocytochemistry shows these proteins are expressed at different levels along the flagellum .

• Developmental timing: CATSPER1 and CATSPER2 transcripts are expressed at different times during spermatogenesis .

• Functional redundancy: Both CATSPER1 and CATSPER2 knockout models show similar phenotypes (failure of hyperactivated motility), suggesting they participate in a common signaling pathway .

• Evolutionary conservation: Comparative studies using antibodies have identified homologs across species, and interesting evolutionary patterns were noted in urochordate Ciona intestinalis, which has ESTs with high homology to CATSPER2 and CATSPER4 .

Recent research suggests CATSPERs may form at least two independent heterotetrameric channels critical to sperm cell motility .

How can calcium imaging be integrated with CATSPER2 immunodetection to study fertility disorders?

Integrating these techniques requires careful methodological considerations:

• Sequential analysis: First perform calcium imaging on live sperm, then fix and process the same samples for CATSPER2 immunodetection.

• Correlative microscopy: Use gridded coverslips to relocate the same cells after processing for immunofluorescence.

• Experimental design for patient studies: In clinical research involving infertile patients with CATSPER2 mutations, perform parallel analyses of:

  • Progesterone-induced calcium responses

  • Ionomycin-induced calcium mobilization

  • CATSPER2 immunolocalization

  • Functional sperm parameters

  • Acrosome reaction assessment

Research has demonstrated that homozygous CATSPER2 deletion affects calcium mobilization in response to both progesterone and ionomycin stimulation, with corresponding effects on the acrosome reaction .