C2CD3 Antibody

What is C2CD3 and why is it significant in research?

C2CD3 is a large protein (canonical form: 2353 amino acids, 260.4 kDa) that functions as a positive regulator of centriole elongation and is critical for primary cilia formation . It contains multiple C2 domains, including PKC-C2 domains with predicted membrane-anchoring functions and divergent C2CD3N-C2 domains . C2CD3 is particularly significant in research because:

• It regulates cilium biogenesis by promoting the assembly of centriolar distal appendages essential for docking ciliary vesicles

• Mutations in C2CD3 cause Orofaciodigital syndrome (OFD14), making it important for understanding ciliopathies

• It serves as an essential regulator of Hedgehog signaling, a critical developmental pathway

• It is widely expressed across tissues with variable isoform patterns, suggesting tissue-specific functions

C2CD3's subcellular localization is primarily in the cytoplasm, specifically at the basal body of cilia .

What are the main applications of C2CD3 antibodies in research?

C2CD3 antibodies have multiple research applications, each with specific methodological considerations:

• Western Blot (WB): Used to detect C2CD3 protein expression and distinguish between isoforms. Typical working concentrations range from 1-2 μg/ml . Researchers should be prepared to detect high molecular weight proteins (205-255 kDa) and optimize separation conditions accordingly .

• Enzyme-Linked Immunosorbent Assay (ELISA): Provides quantitative detection of C2CD3 in complex biological samples .

• Immunohistochemistry (IHC): Visualizes C2CD3 in tissue sections, typically at dilutions of 1:200-1:500 for paraffin-embedded tissues .

• Immunofluorescence (IF): Critical for studying C2CD3's subcellular localization, particularly its association with the basal body of cilia. Often paired with ciliary markers like Arl13b and gamma-tubulin .

When selecting application-specific antibodies, researchers should verify cross-reactivity with their species of interest. Most commercial C2CD3 antibodies are validated for human, mouse, and rat samples .

What are the known isoforms of C2CD3 and how are they distributed across tissues?

C2CD3 exists in multiple isoforms with distinct tissue distribution patterns that researchers should consider when designing experiments:

Up to five different isoforms have been reported in humans . The tissue-specific expression patterns suggest that:

• Researchers should select appropriate positive control tissues based on the isoform of interest

• Experimental outcomes may vary depending on the tissue under investigation

• Antibody selection should consider which isoforms contain the target epitope

These isoform variations contribute to phenotypic variability in C2cd3 mutants and may explain tissue-specific effects of C2CD3 disruption .

How should researchers validate C2CD3 antibody specificity?

Thorough validation of C2CD3 antibodies is essential for generating reliable research data:

• Western blot validation:

  • Verify detection of expected molecular weight bands (primarily 255 kDa, 232 kDa, and 205 kDa)

  • Use gradient gels (3-8%) for proper separation of high molecular weight proteins

  • Include tissue-specific positive controls based on known isoform expression patterns

• Genetic controls:

  • Compare wild-type and C2cd3 mutant or knockdown samples

  • C2cd3 ex2/ex2 mutants should show absence of the 255 kDa band while maintaining the 205 kDa isoform

• Peptide competition:

  • Pre-incubate antibody with the immunizing peptide to confirm specificity

  • Many commercial antibodies offer blocking peptides for this purpose

• Cross-reactivity assessment:

  • Verify antibody performance in the species of interest

  • Consider that protein sequence conservation may vary across species

• Epitope verification:

  • Confirm that the antibody's epitope is present in the isoform or domain of interest

  • For example, antibodies raised against an 18 amino acid peptide near the center of human C2CD3 or against amino acids 1490-1540 should be assessed for which isoforms contain this region

• Functional validation:

  • For immunofluorescence applications, verify correct subcellular localization at the basal body of cilia

  • Co-staining with ciliary markers like Arl13b and gamma-tubulin can confirm proper localization

How can researchers address variability in C2CD3 isoform detection across different tissues?

Effectively addressing tissue-specific variability in C2CD3 isoform detection requires integrated methodological approaches:

• Optimized sample preparation:

  • Use tissue-specific extraction protocols that maintain protein integrity

  • Include protease inhibitors to prevent degradation of high molecular weight isoforms

  • Consider differential centrifugation to separate subcellular fractions where specific isoforms may be enriched

• Gel system optimization:

  • Implement gradient gels (3-8% or 4-15%) for improved separation of high molecular weight proteins

  • Extend running time to achieve better resolution between the 255 kDa, 232 kDa, and 205 kDa isoforms

  • Use wet transfer systems with reduced methanol concentration for efficient transfer of large proteins

• Strategic antibody selection:

  • Use antibodies targeting different epitopes to detect specific isoforms

  • Consider combinations of antibodies targeting N-terminal, C-terminal, and internal regions

  • Verify epitope inclusion in specific isoforms before experimental design

• Tissue-specific positive controls:

  • For detecting the 255 kDa isoform, include embryonic brain or face tissue

  • For the 205 kDa isoform, include heart tissue where it is robustly expressed

  • When available, use recombinant protein standards of known isoforms

• Quantitative analysis:

  • Apply densitometry with appropriate normalization to quantify relative isoform abundance

  • Consider specialized loading controls suitable for high molecular weight proteins

  • Apply statistical approaches that account for tissue-specific variability

By implementing these methodological refinements, researchers can better characterize tissue-specific C2CD3 isoform expression patterns and relate them to functional differences.

What methodological considerations are important when studying C2CD3's role in primary cilia formation?

Investigating C2CD3's role in ciliogenesis requires specialized experimental approaches:

• Imaging methodologies:

  • Implement immunofluorescence co-staining with established ciliary markers:

    • Arl13b for ciliary axoneme visualization

    • Gamma-tubulin for basal body identification

  • Use confocal or super-resolution microscopy for precise localization

  • Consider live-cell imaging to track dynamic ciliary processes

• Genetic manipulation strategies:

  • Utilize available C2cd3 mouse models:

    • Conditional knockout lines for tissue-specific studies

    • CRISPR-targeted lines with mutations in specific domains (C2CD3N-C2 or PKC-C2)

  • Implement domain-specific mutations to dissect structure-function relationships

  • Design rescue experiments with wild-type or mutant constructs

• Ciliary phenotype quantification:

  • Measure cilia frequency, length, and morphology in C2CD3-deficient systems

  • Assess statistical significance with appropriate sample sizes (typically n>100 cells)

  • Evaluate temporal aspects of ciliogenesis through time-course experiments

• Functional readouts:

  • Analyze Hedgehog pathway activity as a functional consequence of ciliary defects

  • Examine Gli protein processing (Gli3FL and Gli3R ratio) in C2CD3 mutants

  • Assess expression of ciliary-dependent genes through qRT-PCR or RNA-seq

• Biochemical interaction studies:

  • Investigate C2CD3's relationship with OFD1, which acts as a negative regulator of C2CD3

  • Perform co-immunoprecipitation to identify interaction partners

  • Consider proximity labeling approaches to identify proteins in close association with C2CD3 at cilia

These methodological approaches enable comprehensive investigation of how C2CD3 contributes to ciliogenesis across different biological contexts.

How does genetic background influence C2CD3 expression and phenotypic manifestations in research models?

Genetic background significantly affects C2CD3 expression patterns and resultant phenotypes, a critical consideration for experimental design:

These data from C2cd3 ex2/ex2 mutants demonstrate that genetic background substantially modifies phenotypic manifestations. Methodological implications include:

• Experimental design considerations:

  • Maintain consistent genetic backgrounds within experimental cohorts

  • Use littermate controls to minimize background-dependent variations

  • Document and report precise genetic background information

• Isoform analysis across backgrounds:

  • The 255 kDa isoform is lost in C2cd3 ex2/ex2 mutants on both C57BL/6J and CD1 backgrounds

  • The 205 kDa isoform is maintained regardless of genetic background

  • Consider background-specific post-translational modifications or interacting partners

• Developmental timing assessments:

  • C2cd3 ex2/ex2 mutants on CD1 background are embryonic lethal at approximately E10.5

  • Monitor developmental progression carefully across different backgrounds

  • Consider background-specific temporal windows for phenotypic analysis

• Translational implications:

  • Background-dependent phenotypic variability in mouse models may parallel genetic modifier effects in human ciliopathies

  • Consider background effects when extrapolating from model systems to human disease

What methodological approaches can resolve discrepancies between predicted and observed molecular weights of C2CD3?

The calculated molecular weight of canonical C2CD3 is approximately 260 kDa , but observed bands can include 255 kDa, 232 kDa, 205 kDa , and even 68 kDa . Resolving these discrepancies requires:

• Technical optimization:

  • Use gradient gels (3-8% or 4-15%) with extended running times

  • Implement specialized transfer techniques for high molecular weight proteins

  • Include high-range molecular weight markers spanning 50-300 kDa

  • Minimize sample heating to prevent aggregation or degradation

• Epitope mapping:

  • Determine if the antibody recognizes:

    • Full-length protein (detecting 255 kDa band)

    • Specific domains present in certain isoforms

    • Processed fragments that retain the epitope

  • Use antibodies targeting different regions to identify specific fragments

• Sample preparation refinement:

  • Test multiple lysis buffers optimized for membrane-associated proteins

  • Include protease inhibitor cocktails to prevent artifactual degradation

  • Compare fresh versus frozen samples to assess storage effects

• Validation through multiple approaches:

  • Confirm band identity through siRNA knockdown or CRISPR knockout

  • Use multiple antibodies targeting different epitopes

  • Compare results with tagged recombinant constructs of known size

• Mass spectrometry validation:

  • Perform immunoprecipitation followed by mass spectrometry to identify protein fragments

  • Analyze post-translational modifications that may affect migration

  • Map identified peptides to determine which protein regions are present

These methodological approaches can help reconcile the discrepancies between predicted and observed molecular weights and improve the reliability of C2CD3 detection in experimental systems.

How can researchers effectively investigate the relationship between C2CD3 and Hedgehog signaling pathways?

Studying C2CD3's role in Hedgehog signaling requires specialized methodological approaches:

• Pathway activation analysis:

  • Measure Hedgehog target gene expression (Gli1, Ptch1) in C2CD3-deficient systems using qRT-PCR

  • Implement Gli-responsive luciferase reporter assays to quantify pathway activity

  • Assess pathway component localization to cilia upon Hedgehog stimulation

• Gli processing assessment:

  • Analyze Gli3 full-length (Gli3FL) to Gli3 repressor (Gli3R) ratio by Western blot

  • C2CD3 mutants often show altered Gli processing due to ciliary defects

  • Use densitometry to quantify processing efficiency across experimental conditions

• Domain-specific function analysis:

  • Generate mutations in specific C2CD3 domains:

    • PKC-C2 domains with predicted membrane-anchoring function

    • Divergent C2CD3N-C2 domains with less understood functions

  • Assess which domains are critical for Hedgehog signaling versus ciliary assembly

  • Determine if specific domains interact with Hedgehog pathway components

• Tissue-specific phenotype analysis:

  • Examine Hedgehog-dependent developmental processes in C2CD3-deficient models:

    • Neural tube patterning (dorsal-ventral axis formation)

    • Limb development (digit patterning)

    • Craniofacial development

• Temporal analysis of signaling:

  • Use inducible systems to manipulate C2CD3 expression at defined developmental stages

  • Determine critical windows when C2CD3 function is essential for Hedgehog response

  • Apply time-lapse imaging to track dynamic changes in signaling component localization

Through these methodological approaches, researchers can comprehensively investigate how C2CD3 contributes to Hedgehog signal transduction and how disruptions in this function contribute to developmental disorders and ciliopathies.