CCDC94 Antibody

What is CCDC94 and what are its primary cellular functions?

CCDC94 (Coiled-coil domain containing 94), also known as YJU2, functions primarily as a splicing factor within the U2-type catalytic step 1 spliceosome. It is required for pre-mRNA splicing both in vivo and in vitro, acting in an ATP-independent manner to promote the first catalytic reaction of pre-mRNA splicing following Prp2-mediated structural rearrangement of the spliceosome . Additionally, CCDC94 plays crucial roles in:

• Negative regulation of DNA damage response

• Signal transduction via p53 class mediator

• Protection of cells from ionizing radiation-induced apoptosis by repressing p53 mRNA expression

The protein contains three predicted coiled-coil domains, which are essential for its functional interactions within the Prp19 complex .

What is the molecular weight and structure of CCDC94?

CCDC94 consists of 323 amino acids with a calculated molecular weight of 37 kDa, though its observed molecular weight in experimental contexts is typically 40 kDa . This discrepancy is likely due to post-translational modifications. The protein is characterized by:

• Three predicted coiled-coil domains that mediate protein-protein interactions

• High evolutionary conservation from yeast to humans, with the first 175 amino acids showing particularly high conservation (94% identity between zebrafish and human)

• UniProt ID: Q9BW85

• GenBank Accession Number: BC000561

Which types of CCDC94 antibodies are available and how should I select one for my experiment?

Various types of CCDC94 antibodies are commercially available with different characteristics:

Selection criteria should include:

• Experimental application (WB, IF, IHC, etc.)

• Target species reactivity

• Desired specificity (monoclonal) vs. epitope coverage (polyclonal)

• Whether post-translational modifications are relevant to your research question

• Clonality and isotype compatibility with your detection systems

What are the recommended dilutions for different experimental applications of CCDC94 antibodies?

Optimal dilutions vary by application and specific antibody. Generally recommended ranges include:

It is strongly recommended to titrate any antibody in your specific experimental system to obtain optimal results, as sensitivity can vary based on sample type, preparation method, and detection system .

How can CCDC94 antibodies be utilized to investigate the role of CCDC94 in DNA damage response pathways?

CCDC94 functions within the DNA double-strand break damage response (DSB-DDR) pathway, making antibodies valuable tools for several approaches:

• Immunoprecipitation coupled with mass spectrometry:

  • To identify CCDC94 interacting partners within the Prp19 complex

  • To characterize protein-protein interactions that change upon DNA damage

• Chromatin immunoprecipitation (ChIP):

  • To assess CCDC94 recruitment to sites of DNA damage

  • To map genomic associations in response to ionizing radiation

• Co-immunostaining with γH2AX:

  • CCDC94 antibodies can be used alongside γH2AX antibodies to investigate localization to DNA damage foci

  • Research has shown that CCDC94 mutants exhibit increased γH2AX positivity, suggesting elevated DNA damage

• Western blotting to quantify p53 expression changes:

  • Following CCDC94 knockdown/knockout

  • After exposure to DNA damaging agents like ionizing radiation

For these applications, recombinant monoclonal antibodies often provide the most consistent results due to their high specificity and batch-to-batch reproducibility .

What controls should be implemented when using CCDC94 antibodies to study R-loop formation and genomic instability?

When investigating R-loops (RNA:DNA hybrids that can lead to genomic instability) in relation to CCDC94:

• Essential negative controls:

  • RNase H treatment of samples prior to antibody application (eliminates genuine R-loops)

  • CCDC94 knockdown/knockout cells to establish baseline signal

  • Isotype-matched control antibodies with no specific target

• Positive controls:

  • Co-staining with S9.6 antibody (recognizes RNA:DNA hybrids directly)

  • Cells treated with splicing inhibitors (which increase R-loop formation)

  • Samples from conditions known to elevate R-loops (e.g., topoisomerase inhibition)

• Validation approaches:

  • qRT-PCR for pre-mRNA vs. mature mRNA ratios to confirm splicing defects

  • DNA-RNA immunoprecipitation (DRIP) assays using S9.6 antibody

  • Cell fractionation to distinguish nuclear vs. cytoplasmic effects

Research has shown that spliceosomal components including CCDC94 protect embryonic neurons from R-loop-associated DNA damage , making these controls critical for accurate interpretation.

What are common technical challenges when working with CCDC94 antibodies and how can they be addressed?

Several technical issues may arise when working with CCDC94 antibodies:

• High background in immunostaining:

  • Increase blocking time (use 5% BSA/0.2% milk/PBS for at least 1 hour)

  • Optimize primary antibody concentration (start with 1:500 dilution for IF)

  • Include additional washing steps with 0.1% Tween-20 in PBS

  • Use cell-type specific positive controls to establish signal specificity

• Multiple bands in Western blot:

  • CCDC94 has an observed molecular weight of 40 kDa

  • Additional bands may represent splice variants, degradation products, or non-specific binding

  • Confirm specificity by knockdown/knockout validation

  • Use fresh samples and add protease inhibitors during extraction

• Low signal strength:

  • Optimize fixation conditions (4% paraformaldehyde for 10-15 minutes works well for IF)

  • For Western blot, load more protein (25-50 μg) and optimize transfer conditions

  • Consider antibody concentration (for Western blot, 1:2000-1:5000 is a good starting range)

  • Use signal enhancement systems appropriate for your detection method

• Fixation-dependent epitope masking:

  • If using paraformaldehyde fixation with poor results, try methanol fixation

  • Some studies recommend dual fixation: 4% PFA for 10 min followed by ice-cold methanol for 5 min at -20°C

How should experimental results be validated to ensure specificity of CCDC94 antibody signal?

To ensure the specificity of signals detected with CCDC94 antibodies:

• Genetic validation:

  • Use CCDC94 knockout or knockdown models (siRNA, CRISPR-Cas9)

  • Rescue experiments with wild-type CCDC94 expression

  • Compare signal in known positive cell lines (e.g., HeLa, HEK-293, Jurkat cells)

• Antibody validation:

  • Use multiple antibodies targeting different epitopes of CCDC94

  • Compare polyclonal and monoclonal antibody results

  • Peptide competition assays to block specific binding

• Signal correlation:

  • Correlate protein expression with mRNA levels using qPCR

  • In mutation studies, confirm the expected phenotypic effects (e.g., increased p53 expression)

• Cross-species validation:

  • CCDC94 is highly conserved across species, so consistent results across human, mouse, and rat samples provide supportive evidence

How can researchers assess CCDC94's role in pre-mRNA splicing using antibody-based techniques?

CCDC94 functions within the spliceosome to facilitate pre-mRNA processing. Several antibody-based approaches can elucidate this function:

• Co-immunoprecipitation with other spliceosome components:

  • Use CCDC94 antibodies to pull down protein complexes

  • Western blot for known Prp19 complex members and spliceosome components

  • Mass spectrometry to identify novel interactions

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq):

  • Map CCDC94 binding sites across the genome

  • Correlate with alternatively spliced exons

  • Compare binding patterns before and after transcriptional inhibition

• Immunofluorescence co-localization with splicing markers:

  • Co-staining with antibodies against SC35 or other splicing speckle markers

  • Analysis of nuclear distribution patterns

  • Observation of changes after treatment with splicing inhibitors

• RNA-immunoprecipitation (RIP):

  • Using CCDC94 antibodies to identify associated RNA species

  • RT-PCR of specific pre-mRNA targets

  • Correlation with splicing efficiency measurements

What methodological approaches can reveal the interplay between CCDC94's splicing functions and p53-mediated apoptosis?

Research has established that CCDC94 represses p53 mRNA expression, thereby protecting cells from ionizing radiation-induced apoptosis . To investigate this relationship:

• Quantitative methods to assess p53 levels:

  • Western blot using both CCDC94 and p53 antibodies in control vs. irradiated cells

  • Flow cytometry with intracellular staining for active caspase-3 following CCDC94 depletion

  • Immunohistochemistry of tissue sections to correlate CCDC94 and p53 expression patterns

• Functional assays:

  • Combine CCDC94 antibody staining with TUNEL assays to correlate protein levels with apoptosis

  • Use live/dead cell discrimination with flow cytometry after manipulating CCDC94 levels

  • Monitor activation of p53 target genes by ChIP following CCDC94 knockdown

• Mechanistic investigation:

  • RNA immunoprecipitation to determine if CCDC94 directly binds p53 mRNA

  • Analysis of p53 pre-mRNA splicing patterns using RT-PCR following CCDC94 manipulation

  • Assessment of p53 mRNA stability in CCDC94-depleted cells

• In vivo models:

  • Immunostaining of zebrafish embryos with CCDC94 and p53 antibodies

  • Analysis of neural tissue development and apoptosis patterns in CCDC94 mutants

  • Rescue experiments using bcl-2 family members to block the apoptotic pathway downstream of p53

What are the optimal storage conditions for preserving CCDC94 antibody activity?

Proper storage is critical for maintaining antibody function:

Most CCDC94 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability during freeze-thaw cycles . For antibodies supplied in PBS only (without stabilizers), aliquoting is strongly recommended to prevent activity loss .

For long-term storage of working dilutions, consider adding BSA (0.1-1%) as a stabilizer . Antibodies with special formulations (e.g., BSA and azide-free versions for conjugation) have specific storage requirements that should be strictly followed .

What validation methods should be applied when testing a new lot of CCDC94 antibody?

When receiving a new antibody lot, consider these validation steps:

• Analytical validation:

  • Western blot against known positive cell lines (HeLa, HEK-293, Jurkat cells are recommended)

  • Look for the expected 40 kDa band

  • Compare signal intensity to previous lots using the same amount of protein

• Functional validation:

  • Immunoprecipitation efficiency comparison

  • Immunofluorescence staining pattern analysis

  • Flow cytometry signal-to-noise ratio assessment

• Cross-reactivity testing:

  • Test against multiple species if the antibody claims cross-reactivity

  • Confirm specificity in knockout/knockdown systems if available

  • Test for non-specific binding in irrelevant cell types

• Application-specific optimization:

  • Establish new working dilutions for each application

  • Determine optimal incubation times and temperatures

  • Document lot-to-lot variations in a laboratory notebook

Recombinant antibody technologies offer advantages in lot-to-lot consistency due to their controlled production methods, reducing the burden of extensive revalidation .

How can CCDC94 antibodies be employed to investigate its role in cancer therapy resistance?

Given CCDC94's role in protecting cells from DNA damage-induced apoptosis, it represents a potential target for enhancing cancer therapy sensitivity:

• Translational research applications:

  • Immunohistochemistry of cancer tissue microarrays to correlate CCDC94 expression with treatment outcomes

  • Comparison of CCDC94 levels before and after radiation or chemotherapy

  • Assessment of nuclear vs. cytoplasmic localization in resistant vs. sensitive tumors

• Mechanistic studies:

  • Co-immunoprecipitation to identify CCDC94 binding partners specific to cancer cells

  • Analysis of p53 pathway activation following CCDC94 inhibition in p53-wild-type cancers

  • Evaluation of R-loop formation and genomic instability upon CCDC94 depletion

• Therapeutic targeting validation:

  • Use antibodies to confirm target engagement of CCDC94 inhibitors

  • Flow cytometry to quantify apoptosis induction following CCDC94 inhibition

  • Combination therapy studies to determine synergy with DNA-damaging agents

Research suggests that pharmacological inactivation of CCDC94 might reduce the threshold for cancer cell apoptotic response, potentially overcoming chemo- and radioresistance .

What approaches can be used to investigate CCDC94's contribution to neurodevelopmental processes?

CCDC94 has been implicated in proper development of the central nervous system, with mutations causing severe sensitivity of embryonic neurons to radiation-induced apoptosis . To study these processes:

• Developmental analysis:

  • Immunohistochemistry to track CCDC94 expression during neural development

  • Double-staining with neuronal markers (e.g., HuC/HuD) to identify affected cell populations

  • Temporal analysis of expression patterns during critical developmental windows

• Functional assessments:

  • Antibody staining following various stressors (oxidative stress, radiation) in neural models

  • Correlation of CCDC94 expression with R-loop formation in developing neurons

  • Investigation of splicing patterns in neuron-specific transcripts

• Pathological studies:

  • Analysis of CCDC94 expression in neurodevelopmental disorders

  • Correlation with markers of genomic instability in patient-derived samples

  • Investigation of potential splicing defects in disease-relevant genes

The zebrafish model has proven particularly valuable for these studies, allowing in vivo analysis of CCDC94 function during neurogenesis through techniques like whole-mount immunohistochemistry and active caspase-3 detection by flow cytometry .

What emerging techniques might enhance the utility of CCDC94 antibodies in research?

Several cutting-edge approaches could expand the applications of CCDC94 antibodies:

• Proximity labeling techniques:

  • APEX2 or BioID fusion constructs with CCDC94 to identify transient interactors

  • Validation of these interactions using conventional co-IP with CCDC94 antibodies

  • Spatial mapping of the CCDC94 interactome in different cellular compartments

• Super-resolution microscopy:

  • Enhanced visualization of CCDC94 within nuclear substructures

  • Co-localization with spliceosome components at nanoscale resolution

  • Dynamic tracking of CCDC94 recruitment to DNA damage sites

• Single-cell approaches:

  • Combining CCDC94 antibody-based detection with single-cell transcriptomics

  • Analysis of heterogeneity in CCDC94 expression and localization

  • Correlation with cell-specific splicing patterns and stress responses

• In situ proximity ligation assays (PLA):

  • Detection of specific CCDC94 protein-protein interactions in fixed cells/tissues

  • Quantification of interaction dynamics following DNA damage

  • Multiplex analysis with other components of the p53 pathway

How might standardized CCDC94 antibody-based assays contribute to personalized medicine approaches?

The role of CCDC94 in modulating DNA damage responses suggests potential clinical applications:

• Predictive biomarker development:

  • Standardized IHC assays to assess CCDC94 expression in tumor samples

  • Correlation with radiation and chemotherapy response

  • Integration into multi-biomarker panels for treatment selection

• Patient stratification:

  • Flow cytometry-based protocols to measure CCDC94 levels in patient samples

  • Establishment of expression thresholds that predict therapy resistance

  • Development of companion diagnostics for CCDC94-targeting therapies

• Treatment monitoring:

  • Serial assessment of CCDC94 expression during treatment

  • Correlation with circulating tumor DNA and other liquid biopsy markers

  • Early detection of resistance development

• Target validation:

  • Antibody-based confirmation of CCDC94 inhibition in clinical trials

  • Pharmacodynamic biomarker development using phospho-specific antibodies

  • Measurement of downstream effects on p53 pathway activation