ccdc149b Antibody

What is CCDC149b and what are its key characteristics in zebrafish models?

CCDC149b (Coiled-Coil Domain Containing 149b) is a protein expressed in zebrafish (Danio rerio) with a calculated molecular weight of approximately 58.3 kDa . It is one of the CCDC family proteins characterized by the presence of coiled-coil domains, which are structural motifs where alpha-helices are coiled together like the strands of a rope. While the specific function of CCDC149b remains largely unknown, researchers utilize antibodies against this protein primarily for detection and characterization studies .

The zebrafish variant of CCDC149 (ccdc149b) is identified in databases under the UniProt accession Q6DH86 and NCBI accession NP_001002710.1 . The protein contains structural regions that can be targeted by different antibodies, with commercially available antibodies typically targeting either the N-terminal region (amino acids 62-89) or other specific domains .

Current research applications focus on using these antibodies for baseline expression studies and potential functional characterization in developmental biology, as zebrafish serve as an important model organism for vertebrate development and disease modeling.

How do antibodies targeting different epitopes of CCDC149 compare in specificity and applications?

The specificity and utility of CCDC149 antibodies vary significantly based on the targeted epitope, with important implications for experimental design:

Antibodies targeting the C-terminal region, such as ABIN2791254, demonstrate broader cross-species reactivity due to higher sequence conservation in this region . In contrast, N-terminal targeting antibodies like ABIN1881144 offer greater species specificity for zebrafish-focused research . The choice between these epitope targets should be dictated by:

• Whether species-specific or cross-species detection is desired

• The structural accessibility of the epitope in your experimental conditions

• The need to distinguish between potential protein isoforms that may lack certain domains

When investigating novel functions or interactions, researchers should consider using antibodies targeting different epitopes to validate findings and rule out epitope-specific artifacts .

What are the critical methodological considerations for Western blot applications using CCDC149b antibodies?

For successful Western blot applications with CCDC149b antibodies, researchers should implement the following methodological approach:

• Sample preparation:

  • For zebrafish samples, optimal extraction requires tissue homogenization in a buffer containing protease inhibitors to prevent degradation of CCDC149b .

  • Complete denaturation is essential due to the coiled-coil structure of the protein; use SDS-PAGE sample buffer with 5% β-mercaptoethanol and heat samples at 95°C for 5 minutes .

• Gel selection and transfer parameters:

  • Use 8-10% polyacrylamide gels for optimal resolution of the ~58 kDa CCDC149b protein .

  • Semi-dry transfer at 15V for 30-40 minutes or wet transfer at 30V overnight at 4°C provides better results for this protein size .

• Blocking and antibody incubation:

  • Blocking with 5% non-fat dry milk is typically sufficient, though 3% BSA may reduce background in some systems .

  • For primary antibody incubation, a 1:1000 dilution is recommended as a starting point, but optimization may be necessary .

  • Extend primary antibody incubation to overnight at 4°C to improve signal strength while maintaining specificity .

• Detection optimization:

  • Enhanced chemiluminescence (ECL) systems are suitable for detection, but longer exposure times may be needed compared to commonly studied proteins .

  • Positive controls from tissues with known CCDC149b expression should be included to validate band specificity .

• Troubleshooting considerations:

  • For weak signals, consider using antibody concentration up to 1:500 and extending development time .

  • Multiple bands may appear due to potential isoforms or post-translational modifications; comparing results with antibodies targeting different epitopes can help identify specific bands .

These methodological refinements are essential given the relatively low expression levels of CCDC149b in many tissues and the structural complexity of coiled-coil domain proteins .

How should researchers optimize storage conditions for CCDC149b antibodies to maintain long-term functionality?

Proper storage of CCDC149b antibodies is critical for maintaining their functionality over time. Based on manufacturer recommendations and empirical evidence, the following storage protocols should be implemented:

For liquid formulation antibodies:

• Short-term storage (up to 1 week): Store at 2-8°C in the original container .

• Long-term storage: Store at -20°C in small aliquots (10-50 μL) to prevent repeated freeze-thaw cycles .

• Add 50% glycerol when creating aliquots for storage below -20°C to prevent freezing damage .

For lyophilized antibodies:

• Upon receipt, store at 4°C until reconstitution .

• Reconstitute using sterile distilled water to the lot-specific concentration indicated .

• After reconstitution, aliquot and store at -20°C, avoiding repeated freeze-thaw cycles .

Additional stability considerations:

• Sodium azide (0.02-0.09%) is typically included as a preservative in these antibodies and helps maintain stability .

• Limit exposure to light, particularly for antibodies stored in clear containers .

• Document the number of freeze-thaw cycles for each aliquot, as performance typically degrades after 3-5 cycles .

Research has shown that antibody activity can remain stable for up to 12-18 months when stored properly at -20°C, though gradual loss of activity may occur over longer periods . For critical experiments, researchers should validate antibody performance prior to use if the storage period exceeds 6 months .

What species cross-reactivity can be expected with different CCDC149 antibodies?

Cross-reactivity profiles vary significantly among CCDC149 antibodies based on epitope conservation across species. This table summarizes expected cross-reactivity patterns:

When evaluating potential cross-reactivity:

• Epitope conservation is the primary determinant: The C-terminal region tends to show higher conservation across species compared to N-terminal regions .

• Validation is essential: Even with high sequence homology, empirical validation is necessary as protein folding and post-translational modifications can affect epitope accessibility across species .

• Cross-reactivity strength varies: Even when cross-reactivity is predicted, signal strength often decreases proportionally with evolutionary distance from the primary target species .

• Applications matter: Cross-reactivity may differ between applications; an antibody showing cross-reactivity in Western blot may not work in immunohistochemistry due to differences in protein conformation and epitope exposure .

Researchers investigating CCDC149 across multiple species should select antibodies targeting more conserved regions or validate multiple antibodies to ensure consistent results .

How can researchers validate the specificity of CCDC149b antibodies in experimental systems?

Validating antibody specificity is critical for generating reliable data. For CCDC149b antibodies, implement these validation approaches:

• Positive and negative control tissues/cells:

  • Use tissues with known CCDC149b expression as positive controls .

  • For zebrafish studies, embryonic tissue at developmental stages with confirmed expression provides an ideal positive control .

  • Use tissues from CCDC149 knockout models (where available) as definitive negative controls .

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide (synthetic peptide corresponding to the target epitope) .

  • A significant reduction in signal indicates specificity for the target epitope .

  • For CCDC149b N-terminal antibodies, use the peptide encompassing amino acids 62-89 for competition assays .

• Multiple antibody verification:

  • Compare results using antibodies targeting different epitopes of CCDC149b .

  • Consistent detection patterns across different antibodies strongly support specificity .

• RNA interference validation:

  • Perform siRNA/morpholino knockdown of CCDC149b in appropriate cell lines or zebrafish embryos .

  • Demonstrate corresponding reduction in the detected protein signal .

• Molecular weight verification:

  • Confirm that the detected band matches the expected molecular weight of ~58 kDa for CCDC149b .

  • Consider isoform variations that might result in bands of different sizes .

• Mass spectrometry confirmation:

  • For advanced validation, immunoprecipitate the protein using the CCDC149b antibody and analyze by mass spectrometry .

  • This provides definitive identification of the captured protein .

These validation steps are particularly important for CCDC149b given the limited characterization of this protein and the potential for cross-reactivity with related coiled-coil domain proteins .

What are the key considerations when using CCDC149 antibodies for immunohistochemistry applications?

When utilizing CCDC149 antibodies for immunohistochemistry (IHC), researchers should consider these methodological aspects to optimize results:

• Fixation and antigen retrieval optimization:

  • CCDC149 antibodies typically require heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) .

  • Optimal fixation is typically 10% neutral buffered formalin for 24-48 hours, as overfixation can mask the CCDC149 epitopes .

  • Paraffin-embedded sections should be cut at 4-6 μm thickness for optimal antibody penetration .

• Antibody selection and dilution:

  • For IHC applications, use antibodies specifically validated for this technique, such as PA561620 or PA560634 .

  • Start with the manufacturer's recommended dilution (typically 1:100 to 1:500) and optimize if necessary .

  • Incubation overnight at 4°C often improves signal-to-noise ratio compared to shorter incubations at room temperature .

• Detection system considerations:

  • Polymer-based detection systems generally provide better results than biotin-streptavidin methods for CCDC149 detection .

  • Signal amplification may be necessary due to potentially low expression levels of CCDC149 .

• Controls and interpretation:

  • Include tissue sections known to express CCDC149 as positive controls .

  • Given the limited characterization of CCDC149 expression patterns, interpretation should be cautious and correlated with other detection methods .

  • Expected cellular localization is primarily cytoplasmic, though nuclear localization has been observed in some cell types .

• Dual staining approaches:

  • For co-localization studies, combine CCDC149 antibodies with markers of cellular compartments or specific cell types .

  • When performing dual immunostaining, sequential rather than simultaneous antibody incubation is recommended to prevent potential interference .

• Counterstaining consideration:

  • Light hematoxylin counterstaining is recommended as heavy counterstaining may obscure low-level CCDC149 signals .

Given the potentially low expression levels of CCDC149 in many tissues, optimizing signal amplification while maintaining specificity is particularly important for successful IHC applications .

How can researchers troubleshoot inconsistent results when using CCDC149b antibodies?

When facing inconsistent results with CCDC149b antibodies, researchers should systematically evaluate and address potential issues:

• Antibody quality and handling issues:

  • Assess antibody degradation through a time-course experiment using positive controls .

  • Check for precipitation in the antibody solution, which may indicate denaturation .

  • For inconsistent results between antibody lots, perform side-by-side comparison with the previously working lot .

• Sample preparation complications:

  • Coiled-coil proteins like CCDC149b can aggregate during preparation; ensure complete denaturation with adequate SDS and heating .

  • Verify protein extraction efficiency by testing multiple extraction protocols (RIPA, NP-40, or urea-based buffers) .

  • For zebrafish samples specifically, ensure developmental stage-appropriate extraction methods as expression may vary during development .

• Technical parameters that affect CCDC149b detection:

  • Optimize transfer conditions for proteins in the 50-60 kDa range, which includes CCDC149b (58 kDa) .

  • Test longer primary antibody incubation times (overnight at 4°C) to improve detection of low-abundance targets .

  • For weak signals, evaluate more sensitive detection systems (enhanced chemiluminescence plus or fluorescent secondary antibodies) .

• Complex result interpretation:

  • Multiple bands may indicate isoforms or post-translational modifications; compare with theoretical molecular weights of known isoforms .

  • Inconsistent band patterns between tissues may reflect biological differences in isoform expression rather than technical issues .

  • For quantification discrepancies, normalize to multiple housekeeping proteins and evaluate linearity of detection .

• Cross-reactivity and specificity concerns:

  • Test antibodies targeting different epitopes of CCDC149b to distinguish specific from non-specific signals .

  • Perform peptide competition assays to confirm specificity of detected bands .

  • Consider potential cross-reactivity with other coiled-coil domain proteins, which share structural similarities .

• Statistical approach to inconsistency:

  • When results vary between experiments, increase biological replicates to determine if variation is biological or technical .

  • Apply appropriate statistical tests that account for technical variability in antibody-based detection methods .

This systematic troubleshooting approach can help resolve inconsistencies commonly encountered with CCDC149b antibodies, particularly given the limited characterization of this protein .

What is known about the expression pattern and potential function of CCDC149 based on antibody studies?

Research using CCDC149 antibodies has provided limited but valuable insights into expression patterns and potential functional implications:

While antibody-based studies have begun to characterize CCDC149 expression, functional studies utilizing these antibodies for techniques like immunoprecipitation, ChIP, or proximity ligation assays would significantly advance understanding of this protein's biological role .

How do pharmacogenomic studies utilize antibodies like anti-CCDC149 for biomarker validation?

In pharmacogenomic research, antibodies against targets like CCDC149 serve crucial roles in validating potential biomarkers identified through genomic screening:

While specific pharmacogenomic applications of CCDC149 antibodies are not extensively documented, the methodological framework for using such antibodies in biomarker validation is well-established in the field .

What are the key differences between monoclonal and polyclonal antibodies targeting CCDC149 in research applications?

When selecting between monoclonal and polyclonal CCDC149 antibodies, researchers should consider these comparative aspects:

Application-specific considerations:

• For Western blotting:

  • Polyclonal CCDC149 antibodies typically provide stronger signals and can detect denatured epitopes effectively .

  • The detection of multiple epitopes makes them more tolerant to partial protein degradation .

• For immunohistochemistry:

  • Polyclonal antibodies like PA561620 and PA560634 are specifically validated for IHC applications .

  • The ability to bind multiple epitopes can improve detection in fixed tissues where some epitopes may be masked .

• For protein interaction studies:

  • For co-immunoprecipitation studies, carefully validated polyclonal antibodies can provide advantages in capturing native protein complexes .

  • When studying protein interactions, researchers should confirm that antibody binding does not interfere with interaction domains .

The predominance of polyclonal antibodies in CCDC149 research likely reflects both the early stage of research on this protein and the practical advantages of polyclonals for initial characterization studies .

How might future antibody development enhance CCDC149 research and potential therapeutic applications?

The advancement of CCDC149 antibody technology will likely progress along several key trajectories that will enhance both basic research and potential therapeutic applications:

• Development of highly specific monoclonal antibodies:

  • Current CCDC149 antibodies are predominantly polyclonal , but future development of monoclonal antibodies would:

    • Provide more consistent reagents with reduced lot-to-lot variability

    • Enable more precise epitope targeting for domain-specific functional studies

    • Support reproducible results across laboratories, addressing current inconsistency challenges in pharmacogenomic studies

• Engineered antibodies with enhanced properties:

  • Similar to therapeutic antibody engineering seen with anti-tau antibodies , CCDC149 antibodies could be engineered for:

    • Reduced Fc core fucosylation to enhance FcγRIIIA binding and cellular effector functions if CCDC149 becomes a therapeutic target

    • Improved tissue penetration for in vivo studies through fragment generation or alternative scaffold development

    • Extended half-life modifications if CCDC149 emerges as a disease-relevant target

• Application-optimized antibody variants:

  • Development of application-specific antibodies for:

    • Super-resolution microscopy to determine precise subcellular localization of CCDC149

    • Proximity labeling approaches to identify interaction partners in living cells

    • Intrabodies for live-cell tracking of CCDC149 dynamics and functional perturbation

• Cross-platform validation systems:

  • Establishment of standardized validation protocols for CCDC149 antibodies would address the inconsistency challenges identified in pharmacogenomic studies :

    • Development of CCDC149 knockout reference samples for definitive specificity testing

    • Creation of standard positive control lysates with defined CCDC149 expression levels

    • Collaborative cross-laboratory validation initiatives similar to those addressing reproducibility in cancer research

• Potential therapeutic applications:

  • If CCDC149 emerges as a disease-relevant target, therapeutic antibody development might follow paths similar to other targets:

    • Antibody-based targeted protein degradation approaches

    • Bispecific antibodies linking CCDC149 to immune effector cells if it becomes an immuno-oncology target

    • Antibody-drug conjugates for targeted delivery if CCDC149 shows cell-type specific expression patterns