CCDC74B Antibody, HRP conjugated

What is CCDC74B protein and why is it studied in research?

CCDC74B (Coiled-coil domain-containing protein 74B) is a human protein encoded by the CCDC74B gene with UniProt accession Q96LY2 . The protein contains coiled-coil structural motifs, which typically facilitate protein-protein interactions and molecular complex formation. While specific functions remain under investigation, coiled-coil domain proteins often participate in cellular processes including structural organization, molecular trafficking, and signal transduction. Research interest in CCDC74B stems from its potential roles in cellular architecture and possible involvement in regulatory pathways that may be relevant to both normal cellular functions and disease states. Detection tools like the HRP-conjugated CCDC74B antibody enable researchers to investigate this protein's expression patterns and cellular localization .

What are the key specifications of commercially available CCDC74B antibody (HRP conjugated)?

The commercially available CCDC74B antibody (HRP conjugated) is a rabbit polyclonal antibody specifically designed to detect human CCDC74B . It has been raised against recombinant human CCDC74B protein (amino acids 1-257) . This antibody has an IgG isotype and is supplied in liquid form with high purity (>95%) after purification by Protein G chromatography . The antibody preparation is formulated in 0.01 M PBS (pH 7.4) containing 0.03% Proclin-300 and 50% glycerol . HRP conjugation enables direct detection in applications like ELISA without requiring secondary antibodies. The product is typically shipped within 5-10 working days and should be stored as aliquots at -20°C, with care taken to avoid repeated freeze/thaw cycles that might compromise antibody activity .

What is the significance of HRP conjugation in CCDC74B antibody applications?

Horseradish peroxidase (HRP) conjugation provides significant methodological advantages in research applications of CCDC74B antibody. This enzyme conjugation enables direct visualization of target binding through enzymatic conversion of various substrates to colored, fluorescent, or chemiluminescent products. In ELISA applications, HRP-conjugated CCDC74B antibody eliminates the need for secondary antibody incubation steps, thereby reducing protocol time, minimizing non-specific binding, and potentially improving sensitivity . The enzymatic amplification provided by HRP allows for detection of even low-abundance CCDC74B protein. Additionally, the HRP conjugation maintains stability under recommended storage conditions (-20°C with 50% glycerol) , ensuring consistent performance across experiments. For research designs requiring maximum sensitivity or when working with limited samples, the direct detection capability of HRP-conjugated antibodies represents a significant methodological advantage over unconjugated primary antibodies.

How should researchers optimize the use of CCDC74B antibody (HRP conjugated) in ELISA applications?

Optimizing CCDC74B antibody (HRP conjugated) for ELISA requires systematic methodology development. Though the antibody is validated for ELISA applications , researchers should first determine optimal working dilutions through titration experiments rather than relying on generic recommendations. A checkerboard titration using serial dilutions of both antigen and antibody will identify conditions yielding maximum signal-to-noise ratio. Critical parameters to optimize include: (1) coating buffer composition and pH; (2) blocking solution formulation (typically 1-5% BSA or non-fat milk); (3) antibody incubation time and temperature; (4) washing buffer composition and number of wash cycles; and (5) substrate selection based on desired sensitivity . When developing multi-analyte assays, cross-reactivity testing is essential. Additionally, researchers should include both positive and negative controls in every experiment to validate results. Given the antibody's high purity (>95%) , non-specific binding should be minimal, but optimization of blocking conditions remains important for maximizing assay specificity, particularly when working with complex biological samples.

How can researchers validate CCDC74B antibody (HRP conjugated) specificity in novel experimental systems?

Validating CCDC74B antibody specificity in novel experimental systems requires a multi-faceted approach beyond manufacturer validation. For definitive specificity confirmation, researchers should implement: (1) Genetic knockdown/knockout validation using RNA interference techniques similar to those described in genome-wide RNAi screening methodologies , where CCDC74B siRNA or shRNA treatment should result in corresponding signal reduction; (2) Overexpression systems where human CCDC74B cDNA transfection should increase signal proportionally; (3) Peptide competition assays using the immunizing peptide (amino acids 1-257 of human CCDC74B) to confirm signal specificity; (4) Western blot analysis to verify detection of a single band at the expected molecular weight; and (5) Cross-species validation if applying the antibody to non-human samples, noting its validated human reactivity . Additionally, researchers should consider using orthogonal detection methods (e.g., immunofluorescence with non-HRP conjugated antibodies) to confirm localization patterns. As CCDC74B contains coiled-coil domains with structural similarities to other proteins, particular attention to potential cross-reactivity with related family members is essential for conclusive experimental interpretation.

What are the considerations for incorporating CCDC74B antibody (HRP conjugated) in multi-parameter flow cytometry experiments?

While CCDC74B antibody (HRP conjugated) is primarily validated for ELISA applications , researchers adapting it for multi-parameter flow cytometry must address several methodological challenges. First, permeabilization protocols must be optimized as CCDC74B is likely intracellular given its coiled-coil domain structure. A systematic comparison of permeabilization reagents (e.g., saponin, methanol, Triton X-100) should be performed to maximize target accessibility while maintaining cellular integrity. Second, the HRP conjugate presents both advantages and limitations in flow cytometry: while it eliminates need for secondary antibodies, spectral overlap with common fluorophores must be carefully evaluated through compensation controls. Researchers should consider the availability of alternative conjugates such as FITC, which is mentioned as an alternative format in the related products section . Third, titration experiments are essential to identify the optimal antibody concentration that maximizes signal-to-noise ratio. Finally, robust controls including fluorescence-minus-one (FMO), isotype controls (rabbit IgG-HRP), and biological controls (CCDC74B-depleted samples) are necessary for accurate data interpretation. Multi-parameter panels should be designed to minimize spectral overlap between the HRP detection channel and other fluorophores used in the analysis.

How does experimental design need to be adapted when investigating post-translational modifications of CCDC74B using the HRP-conjugated antibody?

Investigating post-translational modifications (PTMs) of CCDC74B using HRP-conjugated antibody requires sophisticated experimental design adaptations. First, researchers must determine whether the antibody's epitope (within amino acids 1-257) encompasses or is affected by potential PTM sites. This is critical because PTMs may alter epitope conformation and accessibility. Second, comparative analysis approaches are recommended: parallel immunoprecipitation experiments using both the HRP-conjugated antibody and PTM-specific antibodies (e.g., anti-phospho, anti-ubiquitin) followed by reciprocal detection can confirm modified forms. Third, inhibitor treatments targeting specific modification pathways (phosphatase inhibitors, deubiquitinase inhibitors, etc.) should be incorporated to enrich modified CCDC74B populations. Fourth, subcellular fractionation before immunodetection may be necessary as PTMs often dictate protein localization. Importantly, the HRP conjugation may present methodological limitations for certain PTM detection techniques—researchers should consider using unconjugated CCDC74B antibody versions for applications requiring subsequent probing with PTM-specific antibodies. Finally, mass spectrometry validation of PTM sites following immunoprecipitation with the CCDC74B antibody would provide definitive identification of modification types and locations, complementing antibody-based detection methods.

What approaches can be used to integrate CCDC74B antibody (HRP conjugated) in genome-wide screening methodologies?

Integrating CCDC74B antibody (HRP conjugated) into genome-wide screening methodologies requires sophisticated experimental design drawing from established high-throughput screening principles. Based on RNAi screening methodologies , researchers can develop automated ELISA-based detection systems in 384-well format where the HRP-conjugated antibody serves as the primary readout for CCDC74B expression or localization following systematic genetic perturbations. Critical considerations include: (1) Optimization of reverse transfection protocols in high-density plate formats to achieve consistent gene knockdown efficiency; (2) Development of robust normalization methods using housekeeping controls to account for well-to-well variability; (3) Implementation of Z-factor analysis to ensure assay quality across plates; (4) Integration of image-based detection systems if combining with high-content screening; and (5) Design of bioinformatic pipelines for data analysis similar to those used in genome-wide RNAi screens that incorporate Gene Ontology, Reactome analysis, and STRING network analysis . Notably, researchers should validate hits from primary screens using orthogonal methodologies including qPCR and Western blotting to confirm effects on CCDC74B at both transcript and protein levels. This integrated approach would enable identification of genetic regulators of CCDC74B expression, stability, or post-translational modifications.

What optimization strategies should be employed for Western blot applications of CCDC74B antibody (HRP conjugated)?

While the CCDC74B antibody (HRP conjugated) is primarily validated for ELISA , researchers adapting it for Western blot applications should implement several optimization strategies. First, sample preparation requires careful consideration—complete protein denaturation may affect epitope recognition if the antibody targets conformational epitopes within the coiled-coil domains. Testing both reducing and non-reducing conditions is advisable. Second, transfer efficiency optimization is critical—using PVDF membranes may provide better protein retention than nitrocellulose for coiled-coil domain proteins. Third, blocking conditions require systematic testing; while 5% non-fat milk is standard, BSA-based blockers may yield superior results with HRP-conjugated antibodies by reducing background. Fourth, dilution optimization must be performed empirically, starting with higher concentrations (1:500) and titrating to find optimal signal-to-noise ratio. Fifth, enhanced chemiluminescent (ECL) substrate selection should be based on anticipated expression levels—standard ECL for high abundance or femto-sensitive substrates for low abundance targets. Finally, extended exposure series should be collected to capture the optimal signal window. Including positive controls (human tissue/cell lines with known CCDC74B expression) and negative controls (CCDC74B-knockdown samples) is essential for conclusive interpretation of Western blot results.

How can researchers effectively troubleshoot false negative results when using CCDC74B antibody (HRP conjugated)?

Effectively troubleshooting false negative results with CCDC74B antibody (HRP conjugated) requires systematic methodology assessment. First, verify antibody activity using positive controls known to express human CCDC74B . Second, assess sample integrity through detection of housekeeping proteins in parallel assays. Third, optimize epitope accessibility—for coiled-coil domain proteins like CCDC74B, structural conformations may mask epitopes; test multiple sample preparation methods including different lysis buffers, heat denaturation conditions, and reducing agent concentrations. Fourth, consider detection sensitivity limitations—the standard HRP substrate may be insufficient for low abundance targets; switch to amplified detection systems like tyramide signal amplification. Fifth, evaluate protocol parameters including primary antibody concentration, incubation time/temperature, and buffer compositions—the manufacturer's recommendation should be considered a starting point for optimization rather than definitive conditions . Sixth, check for interfering substances in samples that might inhibit HRP activity; include internal HRP activity controls. Finally, consider expression timing and conditions—CCDC74B expression may be cell-cycle dependent or influenced by culture conditions. For complex samples, enrichment through subcellular fractionation or immunoprecipitation before detection may overcome sensitivity thresholds.

What methodological approaches can determine the cross-reactivity profile of CCDC74B antibody (HRP conjugated) with related coiled-coil domain proteins?

Determining the cross-reactivity profile of CCDC74B antibody requires comprehensive methodological approaches focused on structural and sequence homology assessment. First, researchers should conduct in silico analysis of the immunogen sequence (amino acids 1-257 of human CCDC74B) against protein databases to identify proteins with similar epitope regions, particularly focusing on other coiled-coil domain-containing family members. Second, experimentally validate potential cross-reactivity using a panel of recombinant proteins from identified homologs in ELISA format, comparing binding affinity curves. Third, employ cell lines with differential expression profiles of coiled-coil family members, using RNA-seq data to correlate antibody signal with transcript levels of CCDC74B versus related proteins. Fourth, implement CRISPR-Cas9 knockout systems targeting CCDC74B to create true negative controls; persistent signal in knockout lines would indicate cross-reactivity. Fifth, conduct immunodepletion experiments where sequential immunoprecipitation with CCDC74B antibody should progressively diminish target protein while leaving cross-reactive proteins. Finally, peptide array technology can map the exact epitope recognized by the antibody, enabling precise identification of potentially cross-reactive proteins sharing that specific sequence. This comprehensive approach provides a detailed cross-reactivity profile essential for accurate data interpretation in complex biological systems.

How can CCDC74B antibody (HRP conjugated) be incorporated into studies investigating protein-protein interactions?

Incorporating CCDC74B antibody (HRP conjugated) into protein-protein interaction studies requires methodological adaptation from standard applications. For co-immunoprecipitation experiments, researchers should develop a two-phase approach: first using unconjugated CCDC74B antibody for pulldown followed by detection of interacting partners, then conducting reciprocal experiments using antibodies against suspected interacting proteins followed by detection with the HRP-conjugated CCDC74B antibody. For proximity ligation assays (PLA), the HRP conjugation presents a limitation as this technique typically requires unconjugated primary antibodies; researchers would need alternative CCDC74B antibody formats. In ELISA-based interaction studies, the HRP-conjugated antibody can be effectively employed in sandwich ELISA formats where the interaction partner is captured by its specific antibody, and CCDC74B detection confirms interaction. For functional validation of interactions identified through these methods, researchers should implement genetic approaches (such as those used in genome-wide RNAi screens ) to deplete one partner and observe effects on complex formation. Given that CCDC74B contains coiled-coil domains known to mediate protein-protein interactions, researchers should employ bioinformatic tools like STRING network analysis to predict potential interaction partners based on structural compatibility and co-expression patterns before experimental validation.

What considerations are important when using CCDC74B antibody (HRP conjugated) in tissue microarray analysis for clinical research?

Employing CCDC74B antibody (HRP conjugated) in tissue microarray (TMA) analysis for clinical research requires rigorous methodological considerations. First, fixation and antigen retrieval optimization is critical—researchers should systematically test multiple retrieval methods (heat-induced in citrate, EDTA, or Tris buffers at varying pH) to maximize CCDC74B epitope accessibility in formalin-fixed paraffin-embedded tissues. Second, the HRP conjugation provides a methodological advantage by eliminating secondary antibody steps, but endogenous peroxidase activity in tissues must be thoroughly quenched to prevent false positive signals. Third, titration experiments across different tissue types are essential as optimal antibody concentration may vary between tissues due to differences in protein abundance and matrix effects. Fourth, multi-tissue validation is necessary—the antibody's reactivity to human CCDC74B should be verified across relevant tissue types using positive and negative controls. Fifth, automated staining platforms should be validated against manual protocols to ensure consistency in large-scale TMA studies. Finally, standardized scoring systems must be developed and validated by multiple pathologists, with consideration for both intensity and distribution of staining. For clinical correlation studies, researchers must ensure appropriate statistical power through sample size calculations based on preliminary staining variability data, and implement rigorous quality control measures including periodic re-staining of reference samples to detect any batch effects.

What methodological differences should be considered when comparing data generated using CCDC74B antibody (HRP conjugated) versus genomic detection methods?

When comparing data generated using CCDC74B antibody (HRP conjugated) versus genomic detection methods (e.g., RNA-seq, qPCR), researchers must account for fundamental methodological differences that influence data interpretation. Protein-based detection using the antibody measures post-transcriptional outcomes including translation efficiency, protein stability, and post-translational modifications that genomic methods cannot capture. Several analytical frameworks should be considered: (1) Temporal relationship analysis—protein expression typically lags behind mRNA expression, necessitating time-course studies to establish correlation patterns; (2) Quantitative relationship assessment—mRNA and protein levels often show non-linear relationships due to varying translation rates and protein half-lives; (3) Subcellular localization insights—antibody-based detection can reveal protein compartmentalization information absent in genomic data; (4) Post-translational modification detection—antibody epitopes may be differentially accessible based on modification states, creating apparent discrepancies with transcript levels. To reconcile these methodological differences, researchers should implement integrated analysis approaches incorporating riboprofiling to measure translation efficiency, protein half-life determinations using cyclohexamide chase assays, and correlation analysis across multiple conditions to establish mathematical relationships between transcript and protein levels. This comprehensive approach provides mechanistic insights into CCDC74B regulation beyond what either protein or genomic detection methods can provide independently.

Technical Specifications Table for CCDC74B Antibody (HRP Conjugated)