CCDC93 Antibody

What is CCDC93 and what cellular functions is it associated with?

CCDC93 is a 631-amino acid protein characterized by the presence of a coiled-coil domain, a structural motif crucial for mediating protein-protein interactions. The protein is encoded by a gene located on human chromosome 2, a region associated with several genetic disorders . The coiled-coil domain in CCDC93 facilitates the assembly of protein complexes essential for maintaining cellular functions and responding to environmental signals. CCDC93 plays critical roles in various cellular processes, including gene expression regulation, cell division, and membrane dynamics .

Recent research has uncovered CCDC93's involvement in endosomal trafficking pathways. Studies have linked CCDC93 to the WASH complex and regulation of endosomal trafficking of the copper transporter ATP7A . Additionally, CCDC93 has been implicated in endosomal sorting of Notch receptors through COMMD9-dependent pathways, modulating Notch signaling . These findings suggest CCDC93 participates in complex protein networks governing vesicular transport within cells.

What antibody types are available for CCDC93 detection and their validated applications?

Multiple types of CCDC93 antibodies have been developed for research applications. These include polyclonal antibodies derived from immunization with CCDC93 recombinant protein, such as the rabbit polyclonal antibody described in search result . Monoclonal antibodies are also available, including the mouse monoclonal IgG2a kappa light chain antibody (H-11) that detects CCDC93 protein across multiple species .

The validated applications for CCDC93 antibodies include:

• Western blotting (WB): Used to detect CCDC93 protein in cell and tissue lysates, with observed molecular weight of approximately 73 kDa

• Immunohistochemistry (IHC): Validated for detection in tissues including human colon

• Immunoprecipitation (IP): Effective in isolating CCDC93 protein complexes from cell lysates

• Immunofluorescence (IF): Allows visualization of CCDC93 subcellular localization

• Enzyme-linked immunosorbent assay (ELISA): Provides quantitative assessment of CCDC93 levels

These antibodies demonstrate reactivity across multiple species, including human, mouse, and rat, making them versatile tools for comparative studies .

How should researchers validate CCDC93 antibody specificity before experimental use?

Thorough validation of CCDC93 antibody specificity is essential before proceeding with experiments. A multi-step validation approach should include:

First, western blot analysis should be performed using lysates from multiple cell lines and tissues known to express CCDC93, such as Jurkat cells, human testis tissue, L02 cells, and mouse colon tissue . The antibody should detect a specific band at the expected molecular weight of 73 kDa . Validation should also include negative controls using tissues or cell lines with known low CCDC93 expression.

Second, immunoprecipitation followed by mass spectrometry can confirm antibody specificity. If the antibody specifically pulls down CCDC93, mass spectrometry analysis should identify peptides corresponding to CCDC93 protein sequence . For example, IP using 4 μg of anti-CCDC93 with 1800 μg L02 cell lysate has been validated for specificity .

Third, researchers should consider epitope mapping to determine the specific region of CCDC93 recognized by the antibody. This is particularly important when studying protein interactions or post-translational modifications that might mask the epitope . Finally, knockout or knockdown validation using CCDC93 gene-edited cell lines provides definitive evidence of antibody specificity. Western blotting should show absence or significant reduction of the target band in these models .

What are the optimal dilutions and conditions for using CCDC93 antibodies in different applications?

Optimal working conditions for CCDC93 antibodies vary depending on the specific application:

For Western blotting, the recommended dilution range is 1:500-1:5000 . Starting with a 1:1000 dilution is advisable, with optimization based on signal intensity and background levels. For immunoprecipitation, a dilution range of 1:200-1:2000 is recommended, with 4 μg antibody sufficient for immunoprecipitating CCDC93 from 1800 μg of cell lysate . Immunohistochemistry applications typically require more concentrated antibody, with optimal dilutions ranging from 1:20-1:200 . For paraffin-embedded human colon tissue, a 1:100 dilution has been validated .

Storage conditions are critical for maintaining antibody performance. CCDC93 antibodies should be stored at -20°C in buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, repeated freeze-thaw cycles should be avoided, and manufacturers specifically recommend against aliquoting certain CCDC93 antibody preparations .

For detection systems, both chromogenic and fluorescent secondary antibodies can be used. The H-11 monoclonal antibody is available with various conjugates including horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates, offering flexibility for different detection methods .

How can CCDC93 antibodies be used to investigate protein-protein interactions?

Investigating CCDC93 protein-protein interactions requires sophisticated methodological approaches utilizing specific antibodies. Co-immunoprecipitation (Co-IP) represents a primary technique, where CCDC93 antibodies immobilized on agarose beads can pull down interaction partners from cell lysates . This approach has successfully identified associations between CCDC93 and components of the WASH complex as well as endosomal trafficking machinery .

For studying dynamic interactions, proximity ligation assays (PLA) using CCDC93 antibodies paired with antibodies against suspected interaction partners can visualize protein associations in situ with subcellular resolution. When implementing this approach, researchers should optimize fixation methods to preserve protein complexes while maintaining epitope accessibility.

Advanced approaches include chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) to identify CCDC93-associated protein complexes on a proteome-wide scale. This requires highly specific antibodies validated for immunoprecipitation, such as those mentioned in search result . When designing such experiments, researchers should consider using crosslinking agents to stabilize transient interactions before immunoprecipitation.

For confirming direct interactions, researchers can employ in vitro binding assays using recombinant CCDC93 protein and candidate interactors, with antibodies used for detection via Western blotting or ELISA. This approach helps distinguish direct from indirect interactions identified in cellular contexts.

What is the evidence linking CCDC93 to cardiovascular function and how were antibodies used in these studies?

Recent genetic and functional studies have established a novel role for CCDC93 in cardiovascular physiology. Genome-wide association studies identified a significant association between the rs33975708 variant in CCDC93 and elevated central systolic blood pressure (cSBP), with a strong effect estimate (β = 24.63, SE = 6.62, P = 9.95×10^-8) . This association was consistent across multiple cohorts, suggesting a robust link between CCDC93 genetic variation and cardiovascular phenotypes.

Mechanistic investigations using CCDC93 heterozygous mice (Ccdc93+/-) revealed these animals had 1.3-fold lower aortic Ccdc93 protein expression (P = 0.0041) compared to wild-type littermates, as determined by antibody-based protein quantification . The heterozygous mice exhibited elevated systolic blood pressure (125±10 mmHg vs 110±8 mmHg in controls, P = 0.016), providing a causal link between reduced CCDC93 expression and hypertension .

Wire myography experiments demonstrated that Ccdc93+/- mouse aortae showed impaired acetylcholine-induced relaxation and enhanced phenylephrine-induced contraction, suggesting vascular dysfunction . Additionally, RNA-Seq transcriptome analysis identified significantly enriched pathways related to fatty acid metabolism and mitochondrial function. Western blotting with specific antibodies revealed aberrant expression of mitochondrial proteins Parkin and Nix, pointing to mitochondrial dysfunction as a potential mechanism .

These findings collectively establish CCDC93 as a novel regulator of vascular function and blood pressure, with potential implications for understanding and treating hypertension. CCDC93 antibodies were instrumental in quantifying protein expression differences and characterizing downstream molecular pathways affected by CCDC93 deficiency.

What are common issues encountered when using CCDC93 antibodies and how can they be addressed?

When working with CCDC93 antibodies, researchers may encounter several technical challenges that require systematic troubleshooting approaches:

One common issue is non-specific binding in Western blotting, manifesting as multiple bands or high background. This can be addressed by increasing blocking stringency (5% BSA instead of milk, or addition of 0.1% Tween-20), optimizing antibody dilution (starting with 1:1000 for Western blot), and incorporating additional washing steps . If specificity remains problematic, researchers should consider testing alternative CCDC93 antibodies targeting different epitopes.

For immunohistochemistry applications, antigen retrieval methods significantly impact staining quality. CCDC93 antibodies have been validated for paraffin-embedded tissues with specific dilution requirements (1:100 for human colon tissue) . If signal is weak or absent, optimization of antigen retrieval protocols (heat-induced vs. enzymatic) should be considered. Additionally, signal amplification systems may improve detection sensitivity.

Batch-to-batch variability can affect experimental reproducibility. Researchers should maintain detailed records of antibody lot numbers and perform side-by-side validation when transitioning to new lots. For critical experiments, purchasing sufficient antibody from the same lot is advisable .

Finally, species cross-reactivity should be carefully evaluated. While many CCDC93 antibodies react with human, mouse, and rat proteins, the degree of cross-reactivity may vary. When studying novel species, preliminary validation experiments should confirm antibody reactivity before proceeding with full-scale studies .

How can researchers distinguish between specific and non-specific CCDC93 antibody signals?

Distinguishing specific from non-specific signals requires implementation of rigorous experimental controls and validation approaches:

First, inclusion of appropriate negative controls is essential. These should include: (1) primary antibody omission to identify secondary antibody non-specific binding; (2) isotype controls matching the CCDC93 antibody class (e.g., rabbit IgG for polyclonal antibodies or mouse IgG2a for the H-11 monoclonal antibody) ; and (3) pre-adsorption controls where the antibody is pre-incubated with excess antigen before application to samples.

Competition assays provide strong evidence for specificity. Researchers can perform experiments where increasing concentrations of purified CCDC93 recombinant protein compete with endogenous CCDC93 for antibody binding. Specific signals should decrease proportionally with increasing competitor concentration.

Genetic approaches offer definitive validation. Comparison of samples from wild-type versus CCDC93 knockout or knockdown models should show signal reduction proportional to protein depletion. A heterozygous model (Ccdc93+/-) showed 1.3-fold lower aortic protein expression, providing a useful calibration point for expected signal reduction .

Finally, orthogonal detection methods should show concordant results. If Western blotting, immunofluorescence, and mass spectrometry all indicate CCDC93 presence or absence consistently across sample types, this provides strong evidence for specific detection .

How are CCDC93 antibodies being used to explore the protein's role in disease mechanisms?

CCDC93 antibodies are enabling novel investigations into disease mechanisms, particularly in cardiovascular and metabolic disorders. Recent research has established an important connection between CCDC93 expression and cardiovascular function, with heterozygous Ccdc93+/- mice exhibiting elevated systolic blood pressure, impaired vascular relaxation, and enhanced vasoconstriction . Antibody-based protein quantification confirmed 1.3-fold lower aortic CCDC93 expression in these animals (P = 0.0041), establishing a causal relationship between reduced CCDC93 levels and cardiovascular dysfunction .

Beyond cardiovascular phenotypes, antibody-based studies have revealed connections to metabolic pathways. CCDC93-deficient mice displayed elevated plasma free fatty acid levels (124±13mM vs 96±7mM in controls, P = 0.0031), suggesting disrupted lipid metabolism . Immunoblotting with antibodies against mitochondrial proteins revealed aberrant expression of Parkin and Nix, pointing to mitochondrial dysfunction as a potential disease mechanism .

CCDC93's involvement in endosomal trafficking pathways, particularly for receptors like Notch and transporters like ATP7A, suggests potential implications for developmental and neurological disorders . Ongoing research utilizing specific antibodies is likely to uncover additional disease associations as we better understand CCDC93's diverse cellular functions.

What future technical advances might improve CCDC93 antibody applications in research?

Several technological advances hold promise for enhancing CCDC93 antibody applications in future research. Development of highly specific monoclonal antibodies recognizing distinct epitopes will enable simultaneous detection of different CCDC93 domains or conformations. This would be particularly valuable for studying how protein interactions affect CCDC93 structure and function.

Integration of CCDC93 antibodies with emerging spatial proteomics technologies, such as Imaging Mass Cytometry (IMC) or Multiplexed Ion Beam Imaging (MIBI), will allow simultaneous visualization of CCDC93 alongside dozens of other proteins within tissue microenvironments. Such approaches would provide unprecedented insights into CCDC93's interaction networks in situ.

Advances in nanobody technology may yield smaller, more stable CCDC93-binding reagents that penetrate tissues more effectively and access epitopes inaccessible to conventional antibodies. These could be particularly valuable for studying CCDC93 in complex samples or for super-resolution microscopy applications.

Single-cell proteomics approaches using antibody-based detection will enable analysis of CCDC93 expression and modification heterogeneity across individual cells within tissues. This could reveal previously unappreciated cell-specific functions of CCDC93 relevant to normal physiology and disease states.

Finally, expanding validation across diverse experimental systems using standardized reporting frameworks will improve reproducibility and confidence in CCDC93 antibody-based findings, addressing a critical need in the broader antibody research community .