CRISP2 Antibody

What is CRISP2 protein and what cellular functions is it involved in?

CRISP2 (cysteine-rich secretory protein 2) is a 27 kDa protein primarily expressed in the male reproductive tract. It plays critical roles in sperm function, fertility, and reproductive biology. Research indicates that CRISP2 is involved in sperm-egg fusion processes and may regulate ion channels during fertilization . CRISP2 is also known to interact with prostate secretory protein of 94 amino acids (PSP94), suggesting functional significance in reproductive processes . Expression studies have demonstrated correlations between aberrant CRISP2 expression and fertility issues, including asthenozoospermia and other fertility disorders .

What tissue samples are most appropriate for CRISP2 antibody detection?

CRISP2 antibodies show the strongest reactivity in testis tissue samples across multiple species. Positive Western blot detection has been confirmed in mouse, human, and rat testis tissues . For immunohistochemistry (IHC), human testis tissue provides reliable detection, with recommended antigen retrieval using TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 . When planning experiments, researchers should prioritize these tissue types for optimal detection, particularly when establishing protocols or validating antibody performance.

What applications are CRISP2 antibodies validated for?

Commercial CRISP2 antibodies have been validated for multiple applications, including Western blot (WB), immunoprecipitation (IP), immunofluorescence (IF), immunohistochemistry (IHC), and ELISA . Published research has demonstrated successful applications in knockout/knockdown studies, with at least 7 published applications using Western blot and 2 using immunofluorescence techniques . The consistent detection of the expected 27 kDa band across multiple tissue sources confirms antibody specificity for CRISP2 protein.

What are the optimal dilutions for different applications of CRISP2 antibody?

Based on validated protocols, the following dilutions are recommended for CRISP2 antibody applications:

For optimal results, researchers should titrate the antibody for each specific experimental system and sample type. Different antibody preparations may have varying potencies, necessitating optimization for each new antibody lot .

How should CRISP2 antibodies be stored to maintain efficacy?

CRISP2 antibodies are typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 and should be stored at -20°C . Under these conditions, the antibodies remain stable for at least one year after shipment. For most preparations, aliquoting is unnecessary for -20°C storage, which simplifies laboratory handling protocols. Some preparations (20μl sizes) contain 0.1% BSA as a stabilizer . Researchers should avoid repeated freeze-thaw cycles to maintain antibody performance and specificity over time.

How can I confirm CRISP2 antibody specificity in my experimental system?

Specificity confirmation should employ multiple approaches. First, verify detection of the expected 27 kDa molecular weight band in Western blot applications using testis tissue samples . Second, include appropriate positive controls (testis tissue) and negative controls (tissues not expressing CRISP2). Third, consider using CRISP2 knockout samples as definitive negative controls, as several publications have employed knockout validation techniques . For more rigorous validation, competitive blocking with the immunizing peptide or recombinant CRISP2 protein can demonstrate binding specificity.

How can CRISP2 antibodies be used to investigate male fertility disorders?

CRISP2 expression levels correlate with various fertility parameters. Research has demonstrated that patients with azoospermia, oligoasthenoteratospermia, or asthenospermia exhibit lower CRISP2 expression compared to fertile men . Methodologically, researchers can use CRISP2 antibodies to assess protein expression in sperm samples from infertile patients through Western blot or immunofluorescence techniques. Quantitative analysis should correlate CRISP2 expression with specific parameters such as sperm motility, morphology, and fertilization capacity. Additionally, investigating post-transcriptional regulation of CRISP2 by miR-27b may provide mechanistic insights, as this regulation has been implicated in reduced CRISP2 expression in infertility cases .

What approaches can be used to investigate CRISP2-PSP94 interactions?

CRISP2 has been identified as a PSP94-binding protein in human sperm . To study this interaction, researchers can employ co-immunoprecipitation techniques using CRISP2 antibodies to pull down the protein complex from sperm lysates, followed by Western blot detection of PSP94. Alternatively, the reverse approach using PSP94 antibodies for immunoprecipitation and CRISP2 antibodies for detection can confirm the interaction. For more detailed binding site analysis, peptide competition assays with synthetic peptides corresponding to potential binding regions can help map the interaction domains. Researchers have successfully generated antibodies against specific CRISP2 peptides that do not cross-react with the closely related CRISP3 (which shares 71.4% identity), allowing for specific study of CRISP2-PSP94 interactions .

Can CRISPR/Cas9 be used to develop modified CRISP2 antibodies for site-specific conjugation?

While the search results don't specifically address CRISP2 antibody modification, they do describe a CRISPR/Cas9 genomic editing approach for developing site-specifically modifiable antibodies . This technique could theoretically be applied to CRISP2 antibody-producing hybridomas. The methodology involves using CRISPR/Cas9 to genetically incorporate tags (such as sortase or FLAG tags) at the C-terminal end of the CH3 heavy chain region of the antibody within the hybridoma itself . This genetic modification enables subsequent site-specific conjugation of various cargoes (fluorescent labels, radioactive tags) without compromising antigen binding activity. The advantage of this approach is that it bypasses the need for sequencing variable regions and cloning into producer cell lines, making it more accessible for academic research .

What are common issues when using CRISP2 antibodies in Western blotting and how can they be resolved?

Common issues in Western blotting with CRISP2 antibodies include weak signals, multiple bands, or high background. For weak signals, optimize protein extraction from testis tissue using appropriate lysis buffers containing protease inhibitors to prevent CRISP2 degradation. Increase protein loading (20-50 μg) or adjust antibody concentration within the recommended range (1:500-1:1000) . For multiple bands, verify sample integrity as CRISP2 may undergo post-translational modifications or degradation. Optimize SDS-PAGE conditions and consider using fresh tissue samples. High background can be addressed by extending blocking time (1-2 hours), increasing washing steps, or reducing secondary antibody concentration. When interpreting results, the expected molecular weight for CRISP2 is 27 kDa, and detection should be strongest in testis tissues .

How can I optimize immunohistochemistry protocols for CRISP2 detection in tissue sections?

For optimal IHC detection of CRISP2, use freshly fixed testis tissue sections and test both recommended antigen retrieval methods: TE buffer at pH 9.0 (primary recommendation) and citrate buffer at pH 6.0 (alternative) . Begin with a 1:50 dilution for paraffin-embedded sections, as this has been validated in multiple studies . Implement proper controls, including a no-primary antibody control and positive control (human testis tissue). For challenging samples, extend primary antibody incubation to overnight at 4°C rather than standard 1-2 hours at room temperature. If background is an issue, incorporate an additional blocking step with 5% normal serum from the same species as the secondary antibody. Successful CRISP2 staining should show localization patterns consistent with its expression in developing sperm cells .

How should contradictory CRISP2 expression data from different experimental approaches be reconciled?

When encountering contradictory results between different detection methods (e.g., Western blot versus IHC), consider several methodological factors. First, assess antibody specificity in each application independently, as antibodies may perform differently across techniques due to differences in protein conformation or epitope accessibility. Second, verify consistent tissue processing across experiments, as variation in fixation, extraction methods, or buffer conditions can affect detection. Third, quantify CRISP2 expression using multiple techniques and biological replicates to establish reproducibility. Finally, consult published literature showing successful application in similar experimental contexts . When differences persist, molecular approaches such as mRNA analysis can provide complementary data to protein detection methods to resolve contradictions.

What is known about CRISP2 knockout phenotypes and their implications for reproductive research?

Recent studies with CRISP2 knockout mouse models have provided insights into its physiological relevance. CRISP2 knockout males generated in specific genetic backgrounds showed slight subfertility, suggesting compensatory mechanisms may exist . Unlike other CRISP family members that significantly impact fertility when neutralized by antibodies, immunization with recombinant CRISP2 produced specific antibodies without affecting animal fertility, consistent with its internal localization in sperm . This suggests that while CRISP2 plays a role in sperm function, it may be part of a redundant system. For researchers, these findings indicate that CRISP2 studies should consider genetic background effects and potential compensatory mechanisms when interpreting knockout phenotypes. Multiple fertility parameters should be assessed in knockout models to fully characterize the subtle reproductive effects.

How does post-transcriptional regulation affect CRISP2 expression and function in fertility?

Post-transcriptional regulation, particularly by microRNAs, appears to be a critical mechanism controlling CRISP2 expression in sperm. Studies have identified miR-27b as a regulator of CRISP2 expression, with a correlation between increased miR-27b levels, decreased CRISP2 expression, and reduced sperm motility in infertile patients . For investigating this regulatory mechanism, researchers should employ a comprehensive approach including: (1) quantification of miR-27b and CRISP2 levels in fertile versus infertile samples, (2) in vitro validation using reporter assays to confirm direct targeting, and (3) functional studies examining how modulation of miR-27b affects sperm parameters. This research direction may provide new diagnostic markers for male infertility and potential therapeutic targets for addressing CRISP2-related fertility issues.

What emerging methodologies might enhance CRISP2 antibody applications in reproductive research?

Emerging methodologies that could advance CRISP2 antibody applications include the development of super-resolution microscopy techniques for precise subcellular localization, mass spectrometry-based approaches for identifying CRISP2 interaction partners, and single-cell analysis to understand expression heterogeneity in sperm populations. The CRISPR/Cas9 genomic editing strategy described for antibody modification offers promising avenues for developing sophisticated CRISP2 antibody tools with enhanced functionality, such as site-specific conjugation of fluorescent probes or enzymes. Integration of these advanced approaches with traditional antibody-based detection methods will provide a more comprehensive understanding of CRISP2's role in reproductive biology and potentially identify new therapeutic targets for fertility disorders.