SPEF2 Antibody

What is SPEF2 and why is it important in biomedical research?

SPEF2 (Sperm Flagellar 2) is a protein that plays critical roles in ciliary and flagellar assembly and function. It forms part of the central pair of axonemal components and is highly expressed in the testes . SPEF2 deficiency has been associated with primary ciliary dyskinesia (PCD), affecting approximately 1 in 15,000-20,000 individuals . The importance of SPEF2 in research stems from its involvement in both ciliopathies and male infertility.

Studies have shown that SPEF2 mutations can lead to severe asthenoteratozoospermia due to spermiogenesis failure . Additionally, research on a Japanese patient with a novel SPEF2 variant (c.1860_1861insCT) demonstrated that SPEF2 deficiency can cause PCD with moderate deterioration of ciliary function and reduced numbers of cells with moving cilia . This dual role in reproductive and respiratory systems makes SPEF2 antibodies valuable tools for studying these conditions.

Which tissue types and cellular structures express SPEF2?

SPEF2 is primarily expressed in ciliated structures across multiple tissue types:

• Nasal mucosa cilia and sinus tissues

• Sperm flagella

• Respiratory epithelium

SPEF2 is particularly highly expressed in the testes, which aligns with its critical role in sperm development . Within cells, SPEF2 localizes to the ciliary axoneme, as demonstrated by co-staining with acetylated tubulin (a marker for the entire ciliary axoneme) . Immunofluorescence analysis shows SPEF2 protein appearing as red staining that colocalizes with the green acetylated tubulin staining, resulting in yellow signals in merged images .

How can I verify the specificity of a SPEF2 antibody?

Verifying the specificity of SPEF2 antibodies requires a multi-faceted approach:

• Positive and negative controls: Use tissues known to express SPEF2 (like ciliated respiratory epithelium) as positive controls, and compare with samples from individuals with confirmed SPEF2 mutations. The Japanese patient study effectively used samples from a patient with homozygous SPEF2 mutation showing complete absence of SPEF2 expression .

• Co-staining with ciliary markers: Use established ciliary markers like acetylated tubulin to confirm that SPEF2 antibodies localize to the expected ciliary compartments .

• Western blot analysis: Verify that the antibody detects a protein of the expected molecular weight in wild-type samples, with absence of signal in SPEF2-mutant samples .

• Genetic validation: Correlate antibody staining results with genetic analysis, such as Sanger sequencing to confirm the presence of SPEF2 mutations in negative samples .

• Multiple sample types: Test the antibody on different ciliated tissues to ensure consistent detection patterns.

What are the primary research applications for SPEF2 antibodies?

SPEF2 antibodies have several important research applications:

• Diagnostic confirmation of PCD: Immunofluorescence analysis using SPEF2 antibodies can help confirm PCD cases associated with SPEF2 mutations, as demonstrated in the Japanese patient study .

• Protein interaction studies: Co-immunoprecipitation experiments using SPEF2 antibodies have identified interaction partners including RSPH9 and IFT20, helping to map the molecular networks involved in ciliary assembly .

• Comparative proteomics: SPEF2 antibodies can be used to validate proteomics findings, such as the differential expression of ciliary proteins in SPEF2-mutant samples .

• Developmental studies: Tracking SPEF2 expression during ciliogenesis and sperm development to understand its temporal and spatial regulation.

• Structural analysis: Combined with electron microscopy, SPEF2 antibodies help correlate protein expression with structural features of cilia and flagella .

How can SPEF2 antibodies be used to investigate molecular pathways in primary ciliary dyskinesia?

SPEF2 antibodies enable sophisticated approaches to investigate PCD pathways:

• Interaction network mapping: SPEF2 has been shown to interact with multiple axonemal proteins (SPAG6, RSPH9, SPAG17) and intraflagellar transport proteins (IFT20, IFT27) . SPEF2 antibodies can be used in co-immunoprecipitation experiments to map these interaction networks in different ciliated tissues.

• Comparative proteomics analysis: Research on SPEF2-mutant spermatozoa identified 1,262 differentially expressed proteins (486 upregulated and 776 downregulated) . SPEF2 antibodies can validate these findings in various tissue contexts.

• Functional pathway analysis: By correlating SPEF2 expression with ciliary beat parameters (frequency and amplitude), researchers can determine how SPEF2 deficiency affects specific aspects of ciliary function .

• Structural-functional correlations: The Japanese patient study revealed that despite complete absence of SPEF2 protein, electron microscopy showed no apparent structural abnormalities in cilia . This suggests SPEF2 may primarily affect function rather than structure in respiratory cilia, a hypothesis that can be further investigated using SPEF2 antibodies in functional studies.

What protein interactions does SPEF2 participate in during ciliary assembly?

Research has identified several key SPEF2 protein interactions:

• Radial spoke components: SPEF2 interacts with RSPH9, a component of the radial spoke head that is essential for ciliary motility . This interaction was confirmed by co-immunoprecipitation experiments using FLAG-tagged SPEF2 and HA-tagged RSPH9 .

• Intraflagellar transport proteins: SPEF2 also interacts with IFT20, a component of the intraflagellar transport system essential for ciliary assembly and maintenance .

• Predicted interaction network: STRING analysis predicted SPEF2 interactions with multiple axonemal proteins including SPAG6 and SPAG17, as well as other IFT proteins like IFT27 .

The interaction with both structural components (RSPH9) and transport machinery (IFT20) suggests SPEF2 may serve as a bridge between ciliary structural assembly and the transport system that delivers components to the growing cilium.

How do tissue-specific differences in SPEF2 function affect experimental design?

The search results reveal important tissue-specific differences in SPEF2 function that should inform experimental design:

• Structural vs. functional impacts: In respiratory cilia, SPEF2 deficiency causes moderate functional deterioration without obvious structural abnormalities . In contrast, SPEF2 mutations severely disrupt flagellar assembly in sperm . Experiments should therefore assess both structural and functional parameters.

• Sampling considerations: The Japanese patient study noted that secondary damage from chronic inflammation can affect ciliary analysis . Researchers should consider:

  • Multiple sampling sites (e.g., right and left nasal brushings)

  • Cell culture systems to eliminate inflammatory effects

  • Repeated evaluations to confirm findings

• Protein expression pattern: SPEF2 is highly expressed in testes but may have lower expression in respiratory epithelium . Antibody detection protocols may need to be optimized differently for each tissue type.

• Downstream effector differences: Proteomics analysis of SPEF2-mutant sperm revealed tissue-specific pathways affected by SPEF2 deficiency . Researchers should design experiments to capture these tissue-specific effects.

How can quantitative analysis enhance SPEF2 antibody-based ciliary research?

Quantitative analysis can significantly enhance SPEF2 antibody research in ciliary biology:

• Ciliary beat parameters: In the Japanese patient study, high-speed video microscopy analysis measured ciliary beat frequency (CBF) and ciliary beat amplitude (CBA) . These functional parameters can be correlated with SPEF2 expression levels quantified by immunofluorescence.

• Proportion of ciliated cells: The study noted fewer cells with moving cilia in the SPEF2-mutant patient compared to controls . Quantifying the percentage of SPEF2-positive ciliated cells across samples can reveal patterns in ciliogenesis defects.

• Co-localization coefficients: Mathematical analysis of co-localization between SPEF2 and ciliary markers like acetylated tubulin can provide objective measures of proper protein localization.

• Protein expression levels: Western blot analysis with quantitative densitometry can measure SPEF2 protein levels relative to loading controls or other ciliary proteins .

• Hierarchical clustering analysis: As demonstrated in the proteomic study of SPEF2-mutant sperm, hierarchical clustering with heat maps can reveal patterns in protein expression changes associated with SPEF2 deficiency .

What is the optimal protocol for immunofluorescence staining of SPEF2 in ciliated tissues?

Based on the published research, an optimal immunofluorescence protocol for SPEF2 would include:

• Sample preparation:

  • Nasal brushing samples or surgical sinus tissue specimens

  • Appropriate fixation to preserve epitope accessibility while maintaining tissue architecture

• Antibody and marker selection:

  • Primary antibody: Anti-SPEF2 (such as HPA039606, Atlas antibodies, Bromma, Sweden)

  • Ciliary marker: Anti-acetylated tubulin (T7451, Sigma-Aldrich, St Louis, MO, USA)

  • Nuclear counterstain: 4′,6-diamidino-2-phenylindole (DAPI) (17507, AAT Bioquest, Pleasanton, MO, USA)

• Visualization and analysis:

  • Confocal microscopy to assess co-localization

  • Look for SPEF2 signal (red) colocalizing with acetylated tubulin (green), which appears as yellow in merged images

  • Compare staining patterns between patient samples and appropriate controls

• Controls:

  • Positive control: Ciliated tissue from individuals without SPEF2 mutations

  • Negative control: Samples from individuals with confirmed homozygous SPEF2 mutations

• Validation:

  • Correlate staining results with genetic analysis (e.g., Sanger sequencing of SPEF2)

  • Consider functional correlations using high-speed video microscopy

How can co-immunoprecipitation be optimized for studying SPEF2 protein interactions?

The research describes a specific co-immunoprecipitation approach for studying SPEF2 interactions:

• Expression system setup:

  • HEK293T cells co-transfected with expression vectors encoding:

    • FLAG-tagged SPEF2

    • HA-tagged interaction partners (e.g., IFT20 or RSPH9)

• Cell lysis procedure:

  • Use mammalian protein extraction reagent (Pierce Biotechnology)

  • Supplement with protease inhibitor cocktail (Thermo Fisher Scientific)

  • Follow manufacturer's instructions for complete lysis

• Immunoprecipitation:

  • For FLAG-tagged SPEF2, incubate rabbit polyclonal antibody specific to HA-tagged proteins with protein A/G magnetic beads (20399, Thermo Fisher Scientific)

  • Follow manufacturer's protocol for optimal binding conditions

• Washing and elution:

  • Perform stringent washing to remove non-specific interactions

  • Elute bound proteins with standard 1× sodium dodecyl sulfate (SDS) sample buffer

  • Heat samples for 10 minutes at 100°C prior to analysis

• Detection:

  • Analyze by SDS-PAGE followed by immunoblotting

  • Probe for both FLAG-SPEF2 and HA-tagged interaction partners

  • Include appropriate controls (input lysate, non-specific antibody controls)

This approach successfully verified SPEF2's interaction with both RSPH9 and IFT20 .

How can genetic analysis complement SPEF2 antibody-based research?

Genetic analysis provides crucial complementary information to SPEF2 antibody studies:

• Mutation identification and validation:

  • Whole-exome analysis identified a novel homozygous SPEF2 variant (c.1860_1861insCT) in the Japanese PCD patient

  • Sanger sequencing confirmed the mutation in the patient and heterozygous carrier status in both parents

  • This genetic information enabled interpretation of the immunofluorescence findings showing absence of SPEF2 protein

• Structure-function correlations:

  • Different mutations may affect specific domains of SPEF2

  • Combining genetic data with antibody detection can reveal how mutations affect protein expression, stability, and localization

• Genotype-phenotype correlations:

  • The c.1860_1861insCT mutation resulted in complete absence of SPEF2 protein

  • Despite this, electron microscopy showed no apparent structural abnormalities in cilia

  • This unexpected finding helps define SPEF2's role in ciliary function versus structure

• Family studies:

  • Genetic analysis of the patient's parents revealed heterozygous carrier status

  • This confirms the recessive inheritance pattern of SPEF2-related PCD

  • Antibody studies could potentially explore whether heterozygous carriers show subtle changes in SPEF2 localization or expression

What strategies can improve Western blot detection of SPEF2?

Based on the methodologies described in the research, optimized Western blot detection of SPEF2 should include:

• Sample preparation considerations:

  • For sperm samples: Careful extraction to preserve protein integrity

  • For respiratory tissue: Optimization of lysis conditions to extract membrane-associated proteins

  • Include protease inhibitors to prevent degradation

• Controls and validation:

  • Positive control: Tissues known to express SPEF2 (e.g., normal sperm or ciliated respiratory epithelium)

  • Negative control: Samples from individuals with confirmed SPEF2 null mutations

  • Loading control: Housekeeping protein such as GAPDH or β-actin for normalization

• Detection optimization:

  • Test antibody concentrations to determine optimal signal-to-noise ratio

  • Consider enhanced chemiluminescence for improved sensitivity

  • Use digital imaging systems for quantitative analysis

• Validation approach:

  • The proteomic study verified multiple differentially expressed proteins by Western blot, confirming the proteomics findings

  • This approach can be applied to validate SPEF2 expression changes in different experimental contexts

• Special considerations:

  • SPEF2 is a large protein, so transfer conditions may need optimization

  • If detecting specific domains or isoforms, antibody selection should target appropriate epitopes

How should researchers interpret conflicting results between SPEF2 protein detection and functional assays?

Interpreting conflicting results requires careful consideration of several factors:

What approaches can address antibody specificity challenges in SPEF2 research?

Researchers can address specificity challenges through several strategies:

• Genetic validation:

  • The Japanese patient study demonstrated ideal validation by correlating complete absence of SPEF2 immunostaining with a genetically confirmed homozygous mutation

  • This approach provides definitive confirmation of antibody specificity

• Multi-antibody validation:

  • Use multiple antibodies targeting different SPEF2 epitopes

  • Consistent results across different antibodies increase confidence in specificity

• Blocking peptide controls:

  • Pre-incubate antibody with purified SPEF2 peptide to block specific binding

  • This should eliminate specific staining while non-specific binding remains

• Cross-species validation:

  • Test the antibody in multiple species with conserved SPEF2 sequences

  • Consistent localization patterns increase confidence in specificity

• Combined approaches:

  • Correlate immunofluorescence results with other detection methods

  • For example, the research used both immunofluorescence and Western blotting to assess SPEF2

• Control markers:

  • Always include ciliary markers like acetylated tubulin

  • Specific SPEF2 staining should colocalize with these markers in ciliated structures

How can proteomics approaches enhance SPEF2 antibody research?

Proteomics approaches can significantly enhance SPEF2 antibody research in several ways:

• Comprehensive interaction mapping:

  • Proteomic analysis of SPEF2-mutant spermatozoa identified 1,262 differentially expressed proteins

  • This provides a rich dataset of potential SPEF2-associated proteins for validation with antibody-based methods

• Functional clustering insights:

  • Functional analysis of differentially expressed proteins revealed enrichment for terms such as "spermatid development" and "flagellar assembly"

  • This helps prioritize which protein interactions to validate with SPEF2 antibodies

• Validation of expression changes:

  • The study demonstrated how Western blot analysis with specific antibodies could confirm the expression changes of multiple proteins identified by proteomics

  • This included SPAG6, RSPH1, DYNLT1, TOM20, EFHC1, MNS1 and IFT20

• Heat map visualization:

  • Hierarchical clustering with heat maps revealed consistent patterns of protein expression changes between individuals with SPEF2 mutations

  • Similar approaches can visualize protein networks affected by SPEF2 deficiency

• Identification of novel interactions:

  • STRING analysis predicted multiple SPEF2 interactions with axonemal and IFT proteins

  • These predictions guided successful experimental validation of interactions with RSPH9 and IFT20

What considerations are important when studying SPEF2 in patient samples?

When studying SPEF2 in patient samples, several important considerations should be addressed:

• Secondary ciliary damage:

  • The Japanese patient study noted that chronic inflammation or infection can cause secondary damage to ciliated cells

  • This can complicate interpretation of SPEF2 staining patterns

  • Multiple sampling and/or cell culture models may help mitigate this issue

• Sample heterogeneity:

  • The study observed that "the number of cells with moving cilia was fewer than that in patients without PCD"

  • This highlights the importance of examining sufficient cells in each sample

  • Quantitative approaches should account for this cellular heterogeneity

• Clinical correlation:

  • The Japanese patient had sinusitis and bronchiectasis without situs inversus, and had experienced respiratory symptoms since childhood

  • SPEF2 analysis should be correlated with comprehensive clinical data

  • Pulmonary function test results can provide objective functional parameters

• Control selection:

  • Include appropriate disease controls (e.g., PCD patients with mutations in genes other than SPEF2)

  • Age and sex-matched healthy controls

  • Family members (especially heterozygous carriers) provide valuable comparison

• Ethical considerations:

  • Appropriate informed consent for genetic and protein studies

  • Consideration of incidental findings in whole-exome analyses

  • Responsible reporting of novel variants with uncertain clinical significance