CCDC22 Antibody

What is CCDC22 and why is it significant for research?

CCDC22 is a ubiquitously expressed coiled-coil domain protein involved in multiple cellular functions, including regulation of NF-kappa-B signaling, endosomal recycling of surface proteins, and copper ion homeostasis. It has gained significant research interest due to its association with X-linked intellectual disability (XLID) and its role in the CCC complex, which prevents lysosomal degradation of numerous cargo proteins . Its expression in multiple brain regions, including prefrontal and somatosensory cortex, dentate gyrus, and thalamus, makes it relevant for neuroscience research .

Which species reactivity should be considered when selecting a CCDC22 antibody?

Most commercially available CCDC22 antibodies demonstrate reactivity with human, mouse, and rat samples . Some antibodies show predicted reactivity with additional species based on sequence homology analysis. For example, some CCDC22 antibodies may cross-react with bovine samples due to sequence homology . When selecting an antibody, researchers should verify the validated species reactivity in the product documentation and consider the degree of sequence homology if working with non-validated species .

What are the common applications for CCDC22 antibodies in research?

CCDC22 antibodies are validated for multiple applications including:

• Western Blotting (WB): Typically at dilutions of 1:500-1:2000

• Immunoprecipitation (IP): Using 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Immunocytochemistry/Immunofluorescence (ICC/IF): Validated in various cell types

• Immunohistochemistry (IHC-P): For analysis of CCDC22 expression in tissue sections

• Co-Immunoprecipitation (CoIP): For studying protein-protein interactions

How should I optimize Western blotting protocols for CCDC22 detection?

For optimal Western blot detection of CCDC22 (observed molecular weight: 71-72 kDa), consider these methodological approaches:

• Sample preparation: Use fresh tissues or cells and include protease inhibitors in lysis buffers to prevent degradation

• Gel selection: Use 8-10% gels for optimal resolution around 70 kDa

• Transfer conditions: Employ wet transfer for proteins of this size (71 kDa)

• Blocking: Use 5% BSA in TBST to reduce background (as used in validation studies)

• Antibody dilution: Begin with manufacturer's recommended dilution (typically 1:1000 for WB), then optimize as needed

• Controls: Include positive controls from validated tissues (e.g., human brain tissue, HEK-293 cells) and negative controls where possible

Troubleshooting tip: If multiple bands appear, optimize primary antibody concentration and incubation time, as CCDC22 antibodies may detect degradation products or splice variants.

How can I validate CCDC22 antibody specificity for my experimental system?

Comprehensive validation strategies should include:

• Knockout/knockdown controls: Use CCDC22 knockdown/knockout samples as negative controls, which has been documented in published applications

• Peptide competition assay: Pre-incubate the antibody with immunizing peptide to confirm specificity

• Multiple antibody approach: Compare results using antibodies targeting different epitopes (e.g., N-terminal vs. C-terminal regions)

• Immunogen sequence analysis: Verify the immunogen sequence does not have significant homology with other proteins in your experimental system

• Cross-species validation: If working with non-validated species, perform parallel experiments with validated species samples

How can CCDC22 antibodies be used to investigate the CCC complex and endosomal trafficking?

The CCC complex (containing CCDC22, CCDC93, and C16orf62) regulates endosomal trafficking and recycling of surface proteins. To investigate this complex:

• Co-immunoprecipitation: Use CCDC22 antibodies for CoIP to pull down associated complex members and detect interactions with components like SNX17, retriever complex, and WASH complex proteins

• Confocal microscopy: Perform co-localization studies using CCDC22 antibodies alongside markers for early endosomes, trans-Golgi network, and vesicles to visualize trafficking patterns

• Proximity ligation assay: Detect in situ protein-protein interactions between CCDC22 and other complex components

• Live-cell imaging: Combine with fluorescently tagged trafficking markers to analyze dynamics of CCDC22-positive structures

• Functional assays: Monitor trafficking of known cargo proteins (e.g., integrins ITGA5:ITGB1) in the presence/absence of CCDC22

What methodologies can be used to study CCDC22's role in copper homeostasis?

CCDC22's involvement in copper-dependent ATP7A trafficking between the trans-Golgi network and cell periphery can be studied through:

• Copper challenge experiments: Treat cells with varying copper concentrations and monitor CCDC22-dependent ATP7A localization changes using immunofluorescence

• Proximity labeling approaches: Apply BioID or APEX2 fused to CCDC22 to identify proximal proteins involved in copper homeostasis

• CRISPR/Cas9 gene editing: Create point mutations in CCDC22 domains important for CCC complex formation and assess effects on copper transport

• Quantitative copper measurements: Use inductively coupled plasma mass spectrometry (ICP-MS) to measure cellular copper levels in cells with modified CCDC22 expression

• Structure-function analysis: Generate domain-specific deletions and assess impacts on ATP7A trafficking and copper homeostasis

How can CCDC22 antibodies be utilized to investigate X-linked intellectual disability (XLID)?

CCDC22 has been identified as a candidate gene for syndromic X-linked intellectual disability. For XLID research:

• Patient-derived samples: Compare CCDC22 expression levels in lymphoblast cell lines from XLID patients versus age-matched controls using western blotting and standardized quantification

• Brain region analysis: Use immunohistochemistry with CCDC22 antibodies on post-mortem brain tissue sections to examine expression patterns in regions associated with intellectual disability

• Mutation impact assessment: Generate cell models expressing XLID-associated CCDC22 variants and analyze protein expression, stability, and localization

• Protein interaction network analysis: Perform immunoprecipitation followed by mass spectrometry to identify alterations in CCDC22 protein interactions caused by disease-associated mutations

• Animal models: Validate findings using CCDC22 mutant mouse models that recapitulate XLID features

What approaches can be used to investigate CCDC22's role in NF-κB signaling pathway regulation?

CCDC22 has dual roles in NF-κB signaling - both promoting and inhibiting activation through different mechanisms:

• Stimulus-specific analysis: Compare CCDC22 involvement in different NF-κB activation pathways using pathway-specific stimuli and readouts

• COMMD protein interactions: Investigate differential interactions between CCDC22 and COMMD family proteins (COMMD1 vs. COMMD8) using co-immunoprecipitation and proximity ligation assays

• Ubiquitination studies: Analyze IKBKB ubiquitination and degradation in the presence/absence of CCDC22 or with disease-associated mutations

• CUL-dependent E3 ligase complex analysis: Examine how CCDC22 influences the assembly and activity of CUL1 vs. CUL2-dependent complexes

• Reporter assays: Use NF-κB reporter systems to quantify pathway activity with wild-type vs. mutant CCDC22

How can ChAMP technology be adapted for studying CCDC22 chromatin interactions?

Chromatin Antibody-mediated Methylating Protein (ChAMP) technology offers a novel approach for studying protein-DNA interactions. For CCDC22 research:

• ChAMP adaptation: Utilize the GpC methyltransferase fusion with protein G to tether to CCDC22 antibodies, enabling detection of CCDC22-proximal DNA regions

• Protocol optimization: Adapt fixation conditions (0.1-1% formaldehyde for 5 minutes) followed by heating (65°C for 10 minutes) to maximize enzymatic activity while preserving protein-DNA interactions

• Single-molecule analysis: Apply nanopore sequencing to identify methylation patterns from individual DNA molecules, revealing heterogeneity in CCDC22 binding patterns

• Input minimization: Scale down ChAMP protocols to analyze CCDC22 from limited cell numbers (<100 cells), following post-amplification enrichment strategies

• Multiomic integration: Combine ChAMP-seq data with RNA-seq and proteomics to correlate CCDC22 chromatin associations with transcriptional and protein-level outcomes

What considerations should be made when designing experiments to resolve contradictory findings about CCDC22 function?

When addressing contradictory findings in CCDC22 research:

• Context-dependent activity: Design experiments to test if CCDC22 functions differ across cell types, developmental stages, or disease states

• Isoform-specific analysis: Verify which CCDC22 isoforms are expressed in your experimental system and design isoform-specific detection strategies

• Post-translational modifications: Investigate how phosphorylation or other modifications might switch CCDC22 between activating and inhibitory roles

• Complex composition analysis: Determine how the composition of CCDC22-containing complexes (with different COMMD proteins or cullin-dependent E3 ligases) affects function

• Methodological reconciliation: When contradictory findings emerge, systematically analyze differences in experimental approaches, antibody epitopes, and detection methods