bbof1 Antibody

What is BBOF1 and why is it important in research?

BBOF1, also known as CCDC176 (chromosome 14 open reading frame 45), is a protein containing two coiled-coil domains that is evolutionarily conserved in vertebrates. It functions as a cilia-associated factor induced by the transcription factor foxj1 to mediate the alignment and orientation of cilia in epidermal multi-ciliated cells . BBOF1 has been characterized as essential for sperm flagellar axoneme maintenance and male fertility in mice, with expression elevated during meiosis and accumulation in round spermatids . Its role in ciliary function makes it relevant for research on ciliopathies, reproductive biology, and developmental processes. The 529 amino acid protein has a calculated molecular weight of 62 kDa, which is consistent with observations in experimental systems .

How do I select the appropriate BBOF1 antibody for my research application?

Selecting the appropriate BBOF1 antibody depends on several factors: the species you're working with, your intended application, and specific experimental requirements. For human samples, antibodies with UniProt Primary AC Q8ND07 are appropriate, while mouse samples require antibodies targeting UniProt Primary AC Q3V079 . When choosing an antibody, consider the following criteria:

• Species reactivity: Confirm the antibody reacts with your species of interest (human, mouse, rat)

• Application compatibility: Verify the antibody is validated for your intended application (WB, IHC, ELISA)

• Antigenic region: For different experimental questions, antibodies targeting different regions of BBOF1 may be preferred

• Validation data: Review experimental validation data to ensure performance in conditions similar to your planned experiments

Most commercially available BBOF1 antibodies are polyclonal rabbit antibodies that have been affinity purified and work well for multiple applications .

What are the key differences between detecting BBOF1 in different species?

When working with BBOF1 across different species, researchers should be aware of several important distinctions:

• Protein homology differences: While BBOF1 is conserved across vertebrates, sequence variations exist between human (UniProt ID: Q8ND07) and mouse (UniProt ID: Q3V079) orthologs, requiring species-specific antibodies for optimal detection .

• Expression patterns: BBOF1 shows tissue-specific expression patterns that may vary between species. In mice, BBOF1 is strongly expressed in testis tissue, particularly during meiosis and in round spermatids .

• Cross-reactivity considerations: Some antibodies demonstrate cross-reactivity across human, mouse, and rat samples, but validation within your specific experimental system is essential .

• Molecular weight variations: While the calculated molecular weight for human BBOF1 is 62 kDa, slight variations may exist between species and should be considered when interpreting Western blot results .

• Optimal detection methods: Different fixation and antigen retrieval methods may be required for optimal detection in tissues from different species. For mouse testis tissue, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may be used as an alternative .

What are the recommended protocols for using BBOF1 antibodies in Western blotting?

For optimal Western blot detection of BBOF1, researchers should follow these methodological guidelines:

• Sample preparation:

  • For tissue samples (e.g., mouse or rat testis): Homogenize in RIPA buffer with protease inhibitors, then centrifuge at 14,000g for 15 minutes at 4°C to collect supernatant

  • For cell lines (e.g., TT cells): Lyse cells directly in SDS sample buffer or extract using RIPA buffer with protease inhibitors

• SDS-PAGE separation:

  • Load 20-50 μg of total protein per lane

  • Use 8-10% polyacrylamide gels for optimal separation of the 62 kDa BBOF1 protein

• Antibody incubation:

  • Primary antibody: Dilute BBOF1 antibody 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Secondary antibody: Use appropriate HRP-conjugated secondary antibody (typically anti-rabbit IgG for polyclonal BBOF1 antibodies)

• Detection:

  • Expected molecular weight: 62 kDa for full-length BBOF1

  • Consider using multiple antibodies targeting different regions if studying truncated forms or splice variants

• Validation controls:

  • Positive controls: Mouse testis tissue, TT cells, or rat testis tissue have been validated as positive controls

  • Negative controls: Consider using tissues known to express low BBOF1 levels or BBOF1 knockout samples if available

The protocol may require optimization based on specific sample types and experimental goals.

How should I optimize immunohistochemistry protocols for BBOF1 detection in tissue sections?

For successful immunohistochemical detection of BBOF1 in tissue sections, follow these optimization guidelines:

• Tissue fixation and processing:

  • Use 4% paraformaldehyde fixation for optimal epitope preservation

  • Process tissues using standard paraffin embedding protocols

  • Cut sections at 5-7 μm thickness for optimal antibody penetration

• Antigen retrieval (critical for BBOF1 detection):

  • Recommended method: Heat-mediated antigen retrieval using TE buffer at pH 9.0

  • Alternative method: Citrate buffer at pH 6.0 if TE buffer isn't yielding satisfactory results

  • Heating parameters: 95-98°C for 15-20 minutes followed by gradual cooling

• Blocking and antibody incubation:

  • Block with 5-10% normal serum (matching the species of the secondary antibody) with 1% BSA

  • Dilute BBOF1 antibody between 1:50-1:500, optimizing for your specific tissue

  • Incubate primary antibody overnight at 4°C in a humidified chamber

  • Use appropriate detection system (HRP/DAB or fluorescence-based)

• Controls and validation:

  • Positive control: Mouse testis tissue has been validated for BBOF1 detection

  • Negative controls: Include a no-primary antibody control and, if possible, BBOF1-deficient tissue

  • Consider dual immunofluorescence with known cilia markers to confirm localization

• Signal optimization:

  • If signal is weak, consider increasing antibody concentration or extending incubation time

  • If background is high, increase blocking time/concentration or adjust antibody dilution

  • For specific BBOF1 detection in ciliated cells, consider counterstaining with acetylated tubulin

Methodical optimization of each step will ensure reliable and reproducible BBOF1 detection in tissue sections.

What are the key considerations for setting up BBOF1 ELISA assays?

When establishing ELISA assays for BBOF1 quantification, researchers should consider these methodological aspects:

• Kit selection and setup:

  • Species-specific kits are available for human and mouse BBOF1 detection

  • Test range typically spans 0.156-10 ng/ml, requiring appropriate sample dilution to fall within this range

  • Standard curve preparation: Reconstitute lyophilized standards according to manufacturer instructions and prepare serial dilutions

• Sample preparation:

  • Compatible sample types include tissue homogenates, cell lysates, and biological fluids

  • Tissue homogenization: Use appropriate buffer with protease inhibitors

  • Sample dilution: Perform preliminary experiments to determine optimal dilution factors for your sample type

• Assay optimization:

  • Temperature control: Maintain consistent temperature throughout the procedure

  • Incubation times: Follow recommended times precisely for consistent results

  • Plate washing: Ensure thorough washing between steps to minimize background

• Quality control considerations:

  • Kit stability: Consider that most BBOF1 ELISA kits have 6-month validity with <5% activity loss when stored properly

  • User consistency: Ideally, the same researcher should perform the entire assay to minimize technical variation

  • Batch processing: Process all experimental samples in a single batch when possible to minimize inter-assay variation

• Data analysis:

  • Standard curve fitting: Use appropriate curve-fitting methods (typically 4PL regression)

  • Sample concentration calculation: Account for dilution factors when determining final concentrations

  • Normalization: Consider normalizing to total protein content for tissue/cell samples

For accurate results, sample concentrations must be diluted to mid-range of the kit's detection range, and researchers should be aware that these assays are optimized for native proteins rather than recombinant forms .

How can I investigate BBOF1's role in ciliopathies using antibody-based techniques?

Investigating BBOF1's role in ciliopathies requires sophisticated antibody-based approaches:

• Tissue-specific expression analysis:

  • Perform immunohistochemistry on ciliated tissues from patients with suspected ciliopathies and matched controls

  • Use dual immunofluorescence with BBOF1 antibody (diluted 1:50-1:500) and markers of ciliary components to assess co-localization patterns

  • Consider quantitative image analysis to measure differences in BBOF1 expression levels and subcellular distribution

• Protein-protein interaction studies:

  • Use BBOF1 antibodies for co-immunoprecipitation experiments to identify interacting partners

  • Perform proximity ligation assays (PLA) to visualize and quantify BBOF1 interactions with known ciliary proteins in situ

  • Consider chromatin immunoprecipitation (ChIP) experiments to investigate if BBOF1 associates with specific genomic regions

• Functional analysis in patient-derived cells:

  • Compare BBOF1 expression and localization in primary cells from ciliopathy patients versus controls

  • When studying potential truncation mutations, use antibodies targeting different epitopes of BBOF1 to detect potential truncated forms

  • Combine with nonsense-mediated decay (NMD) inhibitors like cycloheximide to determine if mutations affect mRNA stability

• Mechanistic investigations:

  • Design experiments to measure ciliary function (e.g., beat frequency, intraflagellar transport) while manipulating BBOF1 expression

  • Correlate BBOF1 immunostaining patterns with phenotypic manifestations of ciliopathies

  • Consider studying BBOF1 in the context of known ciliopathy genes such as INTS13, which has been linked to developmental ciliopathies

Recent research has shown that mutations affecting proteins like INTS13 can cause developmental ciliopathies, and gene ontology analysis revealed enrichment in genes involved in cilium biogenesis, suggesting potential interactions with BBOF1 .

What methodological approaches can detect post-translational modifications of BBOF1?

Detecting post-translational modifications (PTMs) of BBOF1 requires specialized antibody-based techniques:

• Phosphorylation analysis:

  • Use phospho-specific antibodies if available, or general phospho-detection methods (Pro-Q Diamond staining)

  • Combine with phosphatase treatment of parallel samples to confirm specificity

  • For mass spectrometry validation, immunoprecipitate BBOF1 using available antibodies (0.3-1 μg/mL concentration) before MS analysis

• Ubiquitination and SUMOylation detection:

  • Perform immunoprecipitation with BBOF1 antibodies followed by Western blotting with anti-ubiquitin or anti-SUMO antibodies

  • Consider using proteasome inhibitors (MG132) in cell culture experiments to stabilize ubiquitinated forms

  • Look for higher molecular weight bands above the expected 62 kDa BBOF1 band

• Glycosylation analysis:

  • Use enzymatic deglycosylation (PNGase F, O-glycosidase) of immunoprecipitated BBOF1

  • Compare migration patterns before and after treatment on Western blots

  • Consider lectin-based detection methods as complementary approaches

• Experimental design considerations:

  • Include appropriate positive controls for each PTM investigation

  • Consider the dynamic nature of PTMs and optimize sample collection and lysis conditions accordingly

  • Compare PTM patterns across different tissues and developmental stages where BBOF1 function is critical

• Data validation:

  • Confirm specificity of PTM detection using site-directed mutagenesis of predicted modification sites

  • Correlate PTM findings with functional assays to determine biological significance

  • Consider computational prediction tools to identify potential PTM sites for targeted investigation

These methodological approaches can provide valuable insights into the regulation of BBOF1 function through post-translational modifications.

How can I study BBOF1 in the context of male infertility research?

To investigate BBOF1's role in male infertility, researchers can employ these specialized antibody-based approaches:

• Expression profiling in spermatogenesis:

  • Perform immunohistochemistry on testis sections using BBOF1 antibodies (1:50-1:500 dilution) to characterize expression patterns during different stages of spermatogenesis

  • Quantify BBOF1 expression in testicular biopsies from men with different forms of infertility compared to fertile controls

  • Co-localize BBOF1 with stage-specific markers of spermatogenesis to precisely determine its temporal expression pattern

• Functional analysis in sperm:

  • Isolate sperm cells from fertile donors and infertile patients for comparative immunofluorescence studies

  • Assess BBOF1 localization in relation to flagellar structures using super-resolution microscopy

  • Correlate BBOF1 expression/localization patterns with sperm motility parameters and other functional measures

• Molecular mechanisms in animal models:

  • Use BBOF1 antibodies validated for mouse and rat samples to study testicular tissues from models with impaired fertility

  • Apply Western blotting (1:500-1:2000 dilution) to quantify BBOF1 levels in wild-type versus mutant models

  • Consider developmental time course studies to track BBOF1 expression throughout sexual maturation

• Genetic variant analysis:

  • Design experiments to detect potential truncated BBOF1 proteins in patients with suspected genetic variants

  • Use antibodies targeting different epitopes to identify potential truncation or missense effects

  • Combine with mRNA stability assays (cycloheximide treatment) to investigate potential nonsense-mediated decay

• Therapeutic potential assessment:

  • Establish experimental systems where BBOF1 expression can be manipulated in cultured cells

  • Monitor effects on cellular phenotypes relevant to sperm formation and function

  • Use antibodies to validate successful BBOF1 manipulation in these experimental systems

Given BBOF1's established role in sperm flagellar axoneme maintenance and male fertility in mice , these approaches can provide valuable insights into its potential contributions to human male infertility.

What are common issues when working with BBOF1 antibodies and how can they be resolved?

Researchers working with BBOF1 antibodies may encounter several technical challenges:

• Weak or absent signal in Western blotting:

  • Problem: Insufficient antibody concentration or protein loading

  • Solution: Increase antibody concentration (try 1:500 instead of 1:2000) , extend incubation time, or increase protein loading

  • Problem: Inefficient protein transfer

  • Solution: Optimize transfer conditions for high molecular weight proteins (~62 kDa)

  • Problem: Degraded protein sample

  • Solution: Use fresh samples, add protease inhibitors, and maintain cold chain

• High background in immunohistochemistry:

  • Problem: Insufficient blocking or antibody cross-reactivity

  • Solution: Extend blocking time, increase blocking agent concentration, and optimize antibody dilution (test range 1:50-1:500)

  • Problem: Inadequate washing

  • Solution: Increase washing duration and volume

  • Problem: Suboptimal antigen retrieval

  • Solution: Test both recommended retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

• Inconsistent ELISA results:

  • Problem: Samples outside the detection range (0.156-10 ng/ml)

  • Solution: Perform preliminary dilution optimization experiments

  • Problem: Temperature fluctuations

  • Solution: Maintain consistent laboratory temperature during assay

  • Problem: Inconsistent technique

  • Solution: Have the same researcher perform the entire assay when possible

• Non-specific bands in Western blot:

  • Problem: Cross-reactivity with related proteins

  • Solution: Increase antibody dilution, extend blocking time, or try alternative antibodies targeting different epitopes

  • Problem: Sample degradation

  • Solution: Use fresh samples with protease inhibitors and analyze band pattern to identify potential degradation products

• Variability between experiments:

  • Problem: Inconsistent storage conditions affecting antibody stability

  • Solution: Aliquot antibodies to avoid freeze-thaw cycles and store according to manufacturer recommendations (typically -20°C with 50% glycerol)

  • Problem: Lot-to-lot variation

  • Solution: Validate each new antibody lot against previous results using consistent positive controls

Methodical optimization and careful attention to experimental variables will help resolve most technical issues encountered when working with BBOF1 antibodies.

How can I validate the specificity of BBOF1 antibodies in my experimental system?

Validating antibody specificity is crucial for generating reliable BBOF1 research data:

• Genetic validation approaches:

  • Use BBOF1 knockout or knockdown models as negative controls

  • Perform rescue experiments by reintroducing BBOF1 into knockout systems

  • Compare staining patterns in tissues known to express varying levels of BBOF1 (high expression in testis tissue)

• Peptide competition assays:

  • Pre-incubate BBOF1 antibody with excess immunizing peptide

  • Run parallel experiments with blocked and unblocked antibody

  • Specific signals should be abolished or significantly reduced in the presence of the blocking peptide

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of BBOF1

  • Compare staining patterns between antibodies targeting different regions (e.g., central versus C-terminal)

  • Consistent patterns across different antibodies suggest specific detection

• Correlative approaches:

  • Compare protein detection with mRNA expression data

  • Perform parallel experiments using complementary methods (e.g., in situ hybridization)

  • Investigate if detected patterns align with known biological functions of BBOF1

• Recombinant protein controls:

  • Express full-length or fragments of BBOF1 as recombinant proteins

  • Use as positive controls in Western blotting

  • Note that antibodies are optimized for native rather than recombinant proteins, which may affect detection efficiency

• Mass spectrometry validation:

  • Immunoprecipitate BBOF1 using available antibodies

  • Analyze by mass spectrometry to confirm the identity of the immunoprecipitated protein

  • This approach can also identify potential interacting partners

These validation approaches should be implemented systematically, particularly when working with new antibody lots or in novel experimental systems.

What are the critical parameters for storage and handling of BBOF1 antibodies?

Proper storage and handling of BBOF1 antibodies is essential for maintaining their performance over time:

• Storage temperature and conditions:

  • Store BBOF1 antibodies at -20°C for long-term stability

  • Most commercial BBOF1 antibodies are supplied in PBS with 50% glycerol and 0.02% sodium azide at pH 7.3, which prevents freezing at -20°C

  • Aliquoting is generally unnecessary for -20°C storage in glycerol-containing buffers, but may be beneficial for frequently used antibodies

• Freeze-thaw considerations:

  • Minimize freeze-thaw cycles to preserve antibody activity

  • For antibodies without glycerol, prepare working aliquots to avoid repeated freezing

  • Allow antibodies to thaw completely at 4°C before use

• Working dilution preparation:

  • Prepare fresh working dilutions for each experiment

  • Dilute in appropriate buffer containing carrier protein (BSA or non-fat dry milk)

  • Gently mix before use to ensure homogeneity

• Contamination prevention:

  • Use sterile technique when handling antibody stock solutions

  • Sodium azide (0.02%) present in storage buffer prevents microbial growth

  • Avoid introducing foreign proteins or microorganisms during handling

• Stability considerations:

  • BBOF1 antibodies are typically stable for one year after shipment when stored properly

  • Some formulations include 0.1% BSA in small (20μl) sizes for additional stability

  • Monitor performance over time by including consistent positive controls in experiments

• Transportation and temporary storage:

  • Transport on ice when moving between laboratories

  • Can be kept at 4°C for short periods (1-2 weeks) during active use

  • Return to -20°C for long-term storage

Adhering to these storage and handling guidelines will help maintain antibody performance and extend the usable life of BBOF1 antibodies.

How can BBOF1 antibodies be used to study ciliopathies and developmental disorders?

BBOF1 antibodies offer powerful tools for investigating ciliopathies and related developmental disorders:

• Ciliopathy diagnostic applications:

  • Use immunohistochemistry with BBOF1 antibodies to analyze patient samples for abnormal BBOF1 expression or localization

  • Develop diagnostic algorithms incorporating BBOF1 staining patterns alongside other ciliary markers

  • Correlate BBOF1 abnormalities with specific ciliopathy phenotypes to improve classification

• Developmental biology investigations:

  • Apply BBOF1 antibodies to study cilia formation and function during embryonic development

  • Track BBOF1 expression across developmental time points in model organisms

  • Investigate potential roles in left-right asymmetry determination, neural tube formation, and other cilia-dependent developmental processes

• Integrative research approaches:

  • Combine BBOF1 antibody-based detection with genetic analysis of ciliopathy-associated genes

  • Study BBOF1 in relation to INTS13, which has been implicated in recessive developmental ciliopathies

  • Use gene ontology analysis to identify other cilia biogenesis genes that may interact with BBOF1

• Translational research applications:

  • Establish patient-derived cell models from ciliopathy patients

  • Use BBOF1 antibodies to characterize ciliary phenotypes in these models

  • Test therapeutic interventions aimed at restoring normal BBOF1 function or compensating for its absence

• Multi-omics integration:

  • Combine immunoprecipitation with BBOF1 antibodies followed by mass spectrometry to identify the BBOF1 interactome

  • Correlate immunostaining data with transcriptomic profiles in normal and pathological contexts

  • Use these integrated datasets to build comprehensive models of BBOF1 function in health and disease

This research direction is particularly promising given recent findings linking ciliary genes to developmental disorders and the established role of BBOF1 in cilia alignment and orientation .

What novel techniques are being developed for studying BBOF1 protein interactions?

Emerging techniques for studying BBOF1 protein interactions are advancing our understanding of its functional networks:

• Proximity-dependent biotinylation approaches:

  • Express BBOF1 fused to BioID or TurboID in relevant cell types

  • Use BBOF1 antibodies to confirm proper localization of fusion proteins

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

  • Validate key interactions using co-immunoprecipitation with BBOF1 antibodies

• Advanced microscopy techniques:

  • Apply super-resolution microscopy (STORM, PALM, SIM) with BBOF1 antibodies to precisely map its subcellular localization

  • Use Förster resonance energy transfer (FRET) or fluorescence lifetime imaging microscopy (FLIM) to detect direct protein interactions in situ

  • Implement live-cell imaging with tagged BBOF1 and validation with antibodies in fixed cells

• Integrative structural biology:

  • Combine antibody epitope mapping with structural prediction algorithms

  • Use cross-linking mass spectrometry (XL-MS) on immunoprecipitated BBOF1 complexes

  • Apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction surfaces

• High-throughput interaction screening:

  • Develop protein arrays probed with BBOF1

  • Use BBOF1 antibodies to detect positive interactions

  • Apply mammalian two-hybrid or split reporter systems with antibody validation

• Computational predictions with experimental validation:

  • Use AI/ML algorithms to predict BBOF1 interaction partners based on structural features

  • Validate predicted interactions through targeted co-immunoprecipitation experiments

  • Confirm specific interactions through reciprocal pulldowns with BBOF1 antibodies

These advanced techniques, when combined with traditional antibody-based methods, provide a comprehensive toolkit for dissecting BBOF1's role in molecular complexes and signaling networks relevant to ciliary function and related disorders.

How might BBOF1 antibodies contribute to understanding evolutionary aspects of ciliary function?

BBOF1 antibodies can provide valuable insights into the evolutionary conservation and divergence of ciliary functions:

• Comparative expression analysis across species:

  • Apply BBOF1 antibodies that recognize conserved epitopes to study expression patterns in diverse vertebrate species

  • Compare subcellular localization in ciliated cells from evolutionarily distant organisms

  • Investigate species-specific variations in expression timing and tissue distribution

• Functional conservation assessment:

  • Use antibodies targeting different regions of BBOF1 to identify structurally conserved domains across species

  • Compare post-translational modification patterns of BBOF1 in different organisms

  • Correlate structural conservation with functional conservation in ciliary orientation and maintenance

• Evolutionary model systems approaches:

  • Apply BBOF1 antibodies to study ciliary structures in evolutionarily informative model organisms

  • Compare BBOF1 localization and function between uniciliated and multiciliated cells across species

  • Investigate potential roles in species with specialized ciliary structures (e.g., sensory cilia, nodal cilia)

• Methodological considerations for cross-species studies:

  • Validate antibody cross-reactivity across target species

  • Optimize fixation and retrieval protocols for each species

  • Consider using multiple antibodies targeting different epitopes to overcome sequence divergence

• Integration with genomic and transcriptomic data:

  • Correlate antibody-based protein detection with genomic conservation analysis

  • Compare BBOF1 expression patterns with transcriptomic profiles across species

  • Use these integrated datasets to reconstruct the evolutionary history of BBOF1 function

This evolutionary perspective can provide important insights into the fundamental aspects of BBOF1 function that have been conserved throughout vertebrate evolution, potentially identifying critical functional domains that could be targeted in therapeutic approaches.