CCDC9 Antibody

What is CCDC9 and what is its biological significance?

CCDC9 is a protein officially known as Coiled-Coil Domain Containing 9, with a calculated molecular weight of approximately 60 kDa (531 amino acids) . It functions primarily as an RNA binding protein, though the full extent of its biological roles is still being investigated . Recent research has identified CCDC9 as a novel candidate gene associated with severe asthenozoospermia, a condition characterized by reduced sperm motility that contributes to male infertility . In a study examining an infertile patient from a consanguineous family, researchers identified a homozygous variant in CCDC9 (NC_000019.9: g.47763960C>T, NM_015603.3, NP_056418.1: p. Ser109Leu) that appears to be highly pathogenic . The protein levels of CCDC9 were significantly reduced in sperm samples containing this variant, with severe damage observed in sperm morphology and ultrastructure .

What are the available types of CCDC9 antibodies for research?

Several types of CCDC9 antibodies are available for research applications:

• Polyclonal antibodies: Examples include rabbit polyclonal antibodies like the one from Proteintech (21104-1-AP), which has been validated for Western blot and ELISA applications, showing reactivity with human and mouse samples .

• Commercial antibodies for immunohistochemistry: Companies like Sigma-Aldrich offer antibodies such as HPA045624, an affinity-isolated antibody that can be used for immunohistochemistry at dilutions of 1:50-1:200 and immunoblotting at 0.04-0.4 μg/mL .

These antibodies are typically generated against specific immunogen sequences of the CCDC9 protein. For instance, the Sigma antibody was developed against the sequence: "IEEDRKKAELEGVAVTAPRKGRSVEKENVAVESEKNLGPSRRSPGTPRPPGASKGGRTPPQQGGRAGMGRASRSWE" .

While the calculated molecular weight of CCDC9 based on its amino acid sequence is approximately 60 kDa, the observed molecular weight in Western blot detection typically ranges from 60-70 kDa . This slight discrepancy between calculated and observed molecular weights is common for many proteins and can be attributed to post-translational modifications, protein folding, or the presence of charged residues that affect migration in SDS-PAGE gels.

How is CCDC9 implicated in male fertility research?

CCDC9 has been identified as a novel candidate gene for severe asthenozoospermia, which is characterized by less than 1% motile sperm in ejaculated semen . Asthenozoospermia affects up to 40% of infertile male patients worldwide, making it the most common cause of male infertility .

In a study of an infertile patient from a consanguineous family, researchers identified a homozygous variant in CCDC9 (p. Ser109Leu) that was predicted to be highly pathogenic through in silico analysis . The study found that:

• CCDC9 protein levels were significantly reduced in sperm samples containing the variant

• Sperm morphology and ultrastructure were severely damaged

• The variant appeared to be associated with the patient's infertility

These findings suggest that CCDC9 plays a crucial role in sperm motility, possibly through structural components of the sperm flagella or related cellular mechanisms . Research into CCDC9's role in sperm function may provide new insights into the molecular basis of male infertility and potential therapeutic targets.

What positive control samples should be used when working with CCDC9 antibodies?

Based on validated experimental data, the following samples have shown positive detection of CCDC9 and can serve as appropriate positive controls:

When designing experiments using CCDC9 antibodies, incorporating these validated positive controls can help ensure proper antibody functionality and experimental reliability. The expression pattern of CCDC9 across these different sample types suggests it may have tissue-specific functions that warrant further investigation .

How should researchers optimize Western blot protocols for CCDC9 detection?

Optimizing Western blot protocols for CCDC9 detection requires careful consideration of several key parameters:

• Sample Preparation:

  • Use appropriate lysis buffers that preserve protein integrity

  • Include protease inhibitors to prevent degradation

  • Denature samples at 95-100°C for 5 minutes in loading buffer containing SDS and a reducing agent

• Gel Electrophoresis:

  • Use 10-12% polyacrylamide gels to effectively resolve the 60-70 kDa CCDC9 protein

  • Include molecular weight markers that cover the 50-75 kDa range

• Antibody Dilution and Incubation:

  • For primary antibody (e.g., Proteintech 21104-1-AP), start with a 1:1000 dilution in 5% BSA or non-fat milk in TBST

  • Optimize by testing a range from 1:500 to 1:3000 as recommended

  • Incubate overnight at 4°C with gentle agitation

• Detection:

  • Use appropriate secondary antibodies (anti-rabbit HRP for most CCDC9 antibodies)

  • Optimize exposure time during chemiluminescent detection to avoid oversaturation

• Special Considerations:

  • When detecting CCDC9 in sperm samples, specialized extraction protocols may be needed due to the high condensation of sperm chromatin and unique protein composition

Following the manufacturer's specific protocols for each antibody is recommended. For example, Proteintech offers a specific Western blot protocol for their CCDC9 antibody (21104-1-AP) .

What methods are available for studying CCDC9 mutations in relation to fertility disorders?

Studying CCDC9 mutations in fertility disorders requires a multifaceted approach:

• Genetic Screening:

  • Whole exome or targeted sequencing to identify variants in the CCDC9 gene

  • Focus on consanguineous families with male infertility cases, which may reveal homozygous variants

  • PCR amplification and Sanger sequencing of CCDC9 exons

• Functional Validation:

  • Western blot analysis to assess CCDC9 protein levels in patient sperm samples

  • Immunofluorescence staining to examine localization and structural abnormalities

  • Sperm motility assays to correlate genetic findings with functional defects

• Ultrastructural Analysis:

  • Electron microscopy to examine sperm flagella structure, mitochondrial sheath, outer dense fibers, and axonemal components

  • Assessment of specific structures including central pair, doublet microtubules, inner dynein arms, and outer dynein arms that may be affected by CCDC9 mutations

• In Silico Analysis:

  • Pathogenicity prediction using computational tools

  • Protein structure modeling to assess the impact of mutations on CCDC9 folding and function

The combination of these approaches provides comprehensive assessment of how CCDC9 variants may contribute to fertility disorders, as demonstrated in research identifying CCDC9 as a candidate gene for severe asthenozoospermia .

How can researchers design immunofluorescence experiments to study CCDC9 localization?

Designing effective immunofluorescence experiments to study CCDC9 localization requires careful attention to several key parameters:

• Sample Preparation:

  • For cultured cells: Fix with 4% paraformaldehyde for 15-20 minutes at room temperature

  • For tissue sections: Use fresh-frozen or properly fixed paraffin-embedded samples

  • For sperm samples: Special fixation protocols may be required to maintain flagellar structure

• Antibody Selection and Validation:

  • Use antibodies validated for immunofluorescence applications

  • Verify specificity using positive controls (HeLa cells, HepG2 cells)

  • Include appropriate negative controls (isotype control or pre-immune serum)

• Protocol Optimization:

  • Test different permeabilization methods (0.1-0.5% Triton X-100, methanol, or saponin)

  • Optimize blocking conditions (3-5% BSA or normal serum)

  • Determine optimal primary antibody dilution (starting with manufacturer recommendations)

  • Include counterstains for cellular landmarks (DAPI for nucleus, phalloidin for actin)

• Co-localization Studies:

  • Consider co-staining with markers of cellular compartments to determine precise localization

  • For sperm studies, include markers for midpiece, mitochondrial sheath, and axonemal components

• Analysis and Quantification:

  • Use confocal microscopy for high-resolution localization analysis

  • Employ image analysis software for quantitative assessment of co-localization coefficients

  • Compare localization patterns between normal samples and those with CCDC9 mutations

These approaches can help establish the subcellular localization of CCDC9 and potentially reveal how mutations affect its distribution and function, particularly in contexts like sperm flagella structure relevant to fertility disorders .

What are common problems in CCDC9 antibody experiments and how can they be addressed?

Researchers working with CCDC9 antibodies may encounter several common challenges:

• Weak or No Signal in Western Blot:

  • Problem: Insufficient protein expression or poor antibody sensitivity

  • Solutions:

    • Increase protein loading (up to 50-80 μg per lane)

    • Reduce antibody dilution (use more concentrated antibody)

    • Extend primary antibody incubation time (overnight at 4°C)

    • Use more sensitive detection systems (enhanced chemiluminescence)

    • Enrich for CCDC9 through immunoprecipitation before Western blot

• Multiple Bands or Non-specific Binding:

  • Problem: Cross-reactivity with other proteins

  • Solutions:

    • Increase blocking time and concentration (5% BSA or milk)

    • Optimize antibody dilution (test range from 1:500-1:3000)

    • Include additional washing steps with higher salt concentration

    • Use freshly prepared buffers to reduce background

    • Consider using a different validated antibody

• Inconsistent Results Across Experiments:

  • Problem: Variable antibody performance or sample preparation

  • Solutions:

    • Aliquot antibodies to avoid freeze-thaw cycles

    • Standardize lysate preparation protocols

    • Include consistent positive controls (HeLa cells, HepG2 cells)

    • Prepare larger batches of buffers to reduce variability

    • Document lot numbers of antibodies used

• Poor Reproducibility in Immunohistochemistry:

  • Problem: Variability in staining intensity or pattern

  • Solutions:

    • Standardize fixation protocols and times

    • Optimize antigen retrieval methods

    • Use automated staining platforms if available

    • Test different antibody dilutions (1:50-1:200 for IHC)

    • Include positive control tissues in each experiment

Careful optimization and standardization of protocols are essential for obtaining reliable results when working with CCDC9 antibodies.

How should researchers interpret variations in CCDC9 molecular weight across different sample types?

Variations in the observed molecular weight of CCDC9 across different sample types can provide valuable biological insights but also present interpretation challenges:

• Expected Molecular Weight Range:

  • The calculated molecular weight of CCDC9 is approximately 60 kDa (531 amino acids)

  • Observed molecular weight typically ranges from 60-70 kDa in Western blot analysis

• Causes of Molecular Weight Variations:

  Potential Cause	Interpretation	Validation Approach
  Post-translational modifications	May indicate functional regulation	Treat samples with phosphatases or glycosidases
  Alternative splicing	May represent tissue-specific isoforms	RT-PCR to identify splice variants
  Protein degradation	May indicate sample quality issues	Include protease inhibitors; fresh sample preparation
  Species differences	May reflect evolutionary changes	Compare with sequence databases

• Sample-Specific Considerations:

  • Cell lines may show different post-translational modification patterns than primary tissues

  • Sperm samples may require specialized extraction conditions that affect protein migration

  • Disease states (e.g., asthenozoospermia) may show altered CCDC9 forms

• Validation Approaches:

  • Use epitope-mapped antibodies to determine if variations affect specific protein regions

  • Employ mass spectrometry to precisely identify modifications or splice variants

  • Compare results from multiple antibodies targeting different epitopes

  • Include recombinant CCDC9 protein as a size reference

How can CCDC9 antibodies be used to investigate disease mechanisms beyond fertility disorders?

While CCDC9 has been primarily studied in the context of male fertility disorders , researchers can leverage CCDC9 antibodies to explore potential roles in other diseases and biological processes:

• Cancer Research:

  • Examine CCDC9 expression across cancer cell lines and patient samples

  • Investigate correlations between CCDC9 expression and clinical outcomes

  • Assess CCDC9 as a potential biomarker using tissue microarrays

• Developmental Biology:

  • Study expression patterns during embryonic development

  • Examine tissue-specific expression in differentiation models

  • Investigate potential roles in cellular specialization

• RNA Biology:

  • Given CCDC9's role as an RNA binding protein , investigate its RNA targets

  • Perform RNA immunoprecipitation followed by sequencing (RIP-seq)

  • Study involvement in RNA processing, transport, or translation

• Neurodegenerative Disorders:

  • Examine expression in neurological tissues

  • Investigate potential roles in RNA metabolism relevant to neurodegeneration

  • Study expression changes in disease models

• Signaling Pathway Analysis:

  • Investigate CCDC9 phosphorylation status in response to stimuli

  • Examine interactions with signaling proteins

  • Study subcellular redistribution upon pathway activation

These research directions would benefit from complementary approaches beyond antibody-based detection, including genetic manipulation (CRISPR/Cas9), transcriptomic analysis, and protein-protein interaction studies to fully elucidate CCDC9's broader biological roles.

What are the latest methodological advances in studying proteins like CCDC9?

Recent technological and methodological advances offer new opportunities for studying CCDC9 and similar proteins:

• Proximity Labeling Techniques:

  • BioID or TurboID fusion proteins to identify proximal interacting partners

  • APEX2-based approaches for temporal analysis of protein neighborhoods

  • Application to identify CCDC9 interaction networks in specific cellular compartments

• Super-Resolution Microscopy:

  • STORM, PALM, or STED microscopy for nanoscale localization studies

  • Multicolor imaging to visualize CCDC9 in relation to cellular structures

  • Live-cell super-resolution to track dynamic behavior

• Cryo-Electron Microscopy:

  • Structural analysis of CCDC9-containing complexes

  • Visualization of CCDC9's role in sperm flagella architecture

  • Comparison between wild-type and mutant structures

• CRISPR-Based Approaches:

  • CRISPR interference or activation to modulate CCDC9 expression

  • Homology-directed repair to introduce specific mutations

  • Base editing to recreate patient mutations in cellular models

• Single-Cell Technologies:

  • Single-cell RNA-seq to assess expression heterogeneity

  • Single-cell proteomics to analyze protein levels across populations

  • Spatial transcriptomics to map expression in complex tissues

• Protein Engineering and Labeling:

  • Split fluorescent protein approaches to study protein-protein interactions

  • HaloTag or SNAP-tag fusions for specific labeling and tracking

  • Optogenetic approaches to control protein function

These advanced methods can be combined with traditional antibody-based approaches to gain deeper insights into CCDC9's structure, function, and role in various biological processes, particularly in contexts where traditional methods have limitations.