CIAO1 Antibody

What is CIAO1 and what are its primary cellular functions?

CIAO1 (also known as CIA1 or WDR39) is an essential component of the cytosolic iron-sulfur (Fe/S) protein assembly machinery. This 38 kDa protein (calculated molecular weight) plays critical roles in several cellular processes:

• Maturation of extra-mitochondrial Fe/S proteins

• Modulation of the trans-activation activity of WT1, potentially regulating cellular growth and differentiation

• Participation in chromosome segregation as part of the mitotic spindle-associated MMXD complex

• Regulation of organ growth through interactions with proteins such as Crumbs (Crb), Galla, and Xpd, as demonstrated in Drosophila models

The protein contains WD repeat domains and is highly conserved across species, indicating its fundamental importance in cellular function. Though its calculated molecular weight is 38 kDa, it is frequently observed at 48-55 kDa in Western blots due to post-translational modifications, particularly phosphorylation .

How do I select the appropriate CIAO1 antibody for my research application?

The selection of an appropriate CIAO1 antibody depends on your specific experimental requirements:

When selecting between monoclonal and polyclonal antibodies:

• Monoclonal antibodies (e.g., PAT6C9A clone) offer high specificity to a single epitope and excellent lot-to-lot consistency

• Polyclonal antibodies can provide higher sensitivity by recognizing multiple epitopes but may have greater batch variation

Always verify species cross-reactivity (human, mouse, rat) in the antibody specifications based on your experimental model .

What are the optimal protocols for using CIAO1 antibodies in Western blot applications?

For optimal Western blot results when detecting CIAO1:

• Sample preparation:

  • Use fresh or properly stored cell/tissue lysates

  • Include protease and phosphatase inhibitors to preserve protein integrity

  • Standardize protein concentration (typically 20-40 μg protein per lane)

• Gel selection and transfer:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • PVDF membranes are recommended for better protein retention

• Antibody incubation:

  • Start with 1:1000 dilution for most CIAO1 antibodies

  • Incubate primary antibody overnight at 4°C for best results

  • For rabbit monoclonal antibodies like CIAO1 (D1B4G), maintain the 1:1000 dilution ratio

• Detection considerations:

  • Expect to detect bands at 48-55 kDa rather than the calculated 38 kDa due to phosphorylation

  • HepG2 and PC-3 cells have been validated as positive controls

  • Include loading controls appropriate for your experimental conditions

• Troubleshooting:

  • If multiple bands appear, consider phosphatase treatment to confirm which bands represent phosphorylated CIAO1

  • For weak signals, extended exposure times or enhanced chemiluminescence substrates may be needed

A detailed standardized protocol improves reproducibility across experiments and allows for more accurate quantitation of CIAO1 expression levels.

How should I design experiments to study CIAO1 interactions with other proteins?

Given CIAO1's role in multiple protein complexes, designing interaction studies requires careful consideration:

• Co-immunoprecipitation approaches:

  • Use antibodies at appropriate dilutions (e.g., 1:200 for IP applications with rabbit mAbs)

  • Consider mild lysis conditions to preserve protein-protein interactions

  • Include appropriate controls (IgG control, input controls)

  • Confirm interactions by reciprocal co-IPs when possible

• GST pull-down assays:

  • Have been successfully used to demonstrate direct binding between CIAO1 and interaction partners like Crb and Xpd

  • Require proper protein expression and purification

  • Can be used to map specific interaction domains

• Proximity ligation assays:

  • Provide spatial resolution of interactions in cellular contexts

  • Particularly useful for visualizing CIAO1 interactions with Fe/S assembly machinery components

• Interaction verification strategies:

  • Genetic interaction studies provide functional validation (as shown with CIAO1 and Xpd in Drosophila)

  • Mutation of key residues can confirm specificity of interactions

  • RNAi-mediated knockdown of potential partners can reveal functional dependencies

When studying CIAO1's interactions with the iron-sulfur cluster assembly complex or with transcription factors like WT1, consider that these interactions may be cell-type specific or condition-dependent .

How can I effectively use CIAO1 antibodies to study its role in iron-sulfur cluster assembly?

CIAO1's critical function in the cytosolic iron-sulfur protein assembly (CIA) machinery makes it a valuable target for investigating Fe/S protein biogenesis:

• Subcellular fractionation approach:

  • Separate cytosolic and mitochondrial fractions to assess CIAO1 distribution

  • Use Western blotting with CIAO1 antibodies (1:1000-1:5000 dilution) to detect location-specific pools

  • Include markers for different cellular compartments as controls

• CIA complex component analysis:

  • Co-immunoprecipitation with CIAO1 antibodies can pull down other CIA components (MMS19, MIP18)

  • Western blotting can confirm interactions and assess complex stability

  • Use 1:200 dilution for immunoprecipitation applications

• Fe/S protein client assessment:

  • Monitor maturation of cytosolic Fe/S proteins following CIAO1 knockdown or overexpression

  • Combine with activity assays for Fe/S-dependent enzymes

  • Use immunofluorescence (1:10-1:100 dilution) to visualize co-localization with client proteins

• Iron sensing and regulation studies:

  • Examine CIAO1 expression under iron deficiency or excess conditions

  • Analyze post-translational modifications using phospho-specific antibodies

  • Correlate CIAO1 levels with iron homeostasis markers

Research has shown that CIAO1 functions in a pathway with Xpd, as evidenced by the fact that knockdown of both CIAO1 and Xpd shows similar phenotypes to knockdown of either gene alone, and that these defects can be bidirectionally rescued through overexpression of the partner protein .

What approaches are recommended for studying CIAO1's role in cell growth and apoptosis regulation?

CIAO1 has been implicated in regulating cell growth and survival pathways, particularly through its interactions with cell cycle regulators and apoptotic machinery:

• Cell proliferation analysis:

  • Use CIAO1 antibodies in immunohistochemistry (1:50-1:500) to correlate expression with proliferative markers in tissue samples

  • Combine with BrdU or EdU labeling to assess S-phase population

  • Analyze CycE levels, as CIAO1 mutant clones show decreased Cyclin E expression

• Apoptosis assessment protocols:

  • CIAO1 knockdown has been shown to increase apoptotic cell death

  • Use TUNEL assays or caspase activity measurements in conjunction with CIAO1 antibody staining

  • Monitor Diap1 (death-associated inhibitor of apoptosis 1) levels, which are reduced in CIAO1 mutant cells

• Genetic interaction experiments:

  • Overexpression of Diap1 in CIAO1 mutant clones induces CycE expression, suggesting that reduced CycE is secondary to loss of Diap1

  • CycE overexpression can restore growth defects from CIAO1 RNAi

  • Design rescue experiments based on these pathway relationships

• Organ growth model systems:

  • In Drosophila, CIAO1 interacts with Crumbs and Xpd to regulate organ growth

  • Imaging studies using immunofluorescence (1:10-1:100) can detect these interactions in situ

  • Analyze phenotypes in CIAO1 mutant clones versus complete tissue knockout

A detailed quantitative analysis of cell cycle progression, apoptotic markers, and tissue growth parameters should be included in these experiments to fully characterize CIAO1's role in balancing proliferation and cell death.

How can I resolve discrepancies in CIAO1 molecular weight detection?

The discrepancy between CIAO1's calculated molecular weight (38 kDa) and its observed weight (48-55 kDa) is a common source of confusion:

• Post-translational modification analysis:

  • The higher observed molecular weight is primarily attributed to phosphorylation

  • Perform lambda phosphatase treatment of samples prior to SDS-PAGE to confirm

  • Run treated and untreated samples side-by-side to visualize mobility shift

• Protocol optimization for consistent detection:

  • Use gradient gels (4-15%) to better resolve the phosphorylated forms

  • Optimize transfer conditions for higher molecular weight proteins

  • Consider using PVDF membranes which may retain the phosphorylated forms better than nitrocellulose

• Antibody selection considerations:

  • Some antibodies may preferentially recognize particular phosphorylated states

  • Verify which forms your selected antibody detects by consulting validation data

  • Consider using multiple antibodies targeting different epitopes for comprehensive analysis

• Experimental controls:

  • Include positive controls where CIAO1 expression has been validated (HepG2, PC-3 cells)

  • Run samples from CIAO1 knockdown or knockout systems as negative controls

  • Use recombinant CIAO1 protein as a size reference when available

Understanding these variations is crucial for accurate data interpretation, especially when comparing results across different experimental conditions or antibody sources.

What are the critical variables in optimizing immunohistochemistry protocols for CIAO1 detection?

Successful immunohistochemical detection of CIAO1 requires careful optimization of multiple parameters:

• Fixation and antigen retrieval:

  • CIAO1 detection in tissues often requires specific antigen retrieval methods

  • TE buffer at pH 9.0 is suggested as the primary retrieval method

  • Citrate buffer at pH 6.0 can be used as an alternative approach

  • Optimization of retrieval time and temperature is critical

• Antibody selection and dilution:

  • Start with recommended dilutions (1:50-1:500) and optimize based on signal-to-noise ratio

  • Polyclonal antibodies may provide better sensitivity in IHC applications

  • Validate specificity using tissues known to express CIAO1 (e.g., human small intestine, testis)

• Detection system considerations:

  • Polymeric detection systems often provide better sensitivity than avidin-biotin methods

  • Tyramide signal amplification can be employed for detecting low abundance CIAO1

  • Chromogen selection affects contrast and compatibility with counterstains

• Multiplex immunostaining strategies:

  • For co-localization studies with other proteins (e.g., WT1, cell cycle markers)

  • Sequential staining protocols may be necessary to avoid antibody cross-reactivity

  • Consider spectral imaging for distinguishing multiple stains

• Validation and controls:

  • Include positive control tissues with known CIAO1 expression

  • Use CIAO1 knockdown or knockout tissues as negative controls

  • Implement no-primary-antibody controls to assess non-specific binding

These optimizations are particularly important when studying CIAO1's expression in relation to tissue growth, differentiation, and pathological conditions.

How should researchers interpret changes in CIAO1 expression in the context of cell cycle regulation?

Interpreting CIAO1 expression in relation to cell cycle requires consideration of several interconnected pathways:

• CIAO1-Cyclin E relationship:

  • CIAO1 mutant clones show decreased Cyclin E (CycE) levels

  • This reduction appears to be secondary to loss of Diap1, as Diap1 overexpression in CIAO1 mutant clones induces CycE expression

  • Analyze both proteins when assessing CIAO1's impact on cell cycle

• Cell cycle progression analysis:

  • Correlate CIAO1 expression with cell cycle phase markers

  • Flow cytometry combined with CIAO1 immunostaining can reveal phase-specific relationships

  • Time-lapse microscopy with fluorescently tagged CIAO1 can track dynamic changes during cycle progression

• Quantitative assessment guidelines:

  • Use automated image analysis for unbiased quantification of CIAO1 and cell cycle marker levels

  • Present data as scatter plots showing cell-to-cell variation rather than simple averages

  • Statistical analysis should account for cell cycle phase distribution in the population

• Multi-parameter data integration:

  • Consider CIAO1's role in Fe/S protein maturation when interpreting cell cycle effects

  • Analyze both nuclear and cytoplasmic CIAO1 localization, as these pools may have distinct functions

  • Correlate with chromosome segregation efficiency, given CIAO1's role in this process

When interpreting CIAO1 knockdown phenotypes, consider that CycE overexpression is sufficient to restore growth defects from CIAO1 RNAi, positioning CycE as a downstream effector in CIAO1-mediated growth regulation .

What experimental approaches can resolve contradictory findings about CIAO1's role in different cellular contexts?

Resolving conflicting data about CIAO1 function requires systematic approaches:

• Cell type-specific analysis:

  • CIAO1 functions may vary between cell types due to different interaction partners

  • Compare results across multiple validated cell lines (e.g., HepG2, PC-3)

  • Use conditional knockout models to assess tissue-specific effects in vivo

• Contextual interaction mapping:

  • CIAO1 interacts with multiple partners (Crb, Galla, Xpd, WT1)

  • Determine which interactions predominate under specific conditions

  • Use proximity labeling techniques (BioID, APEX) to create unbiased interaction maps

• Resolution of functional redundancy:

  • Test for compensatory mechanisms by other CIA components

  • Perform simultaneous knockdown of CIAO1 and related proteins

  • Analyze subtle phenotypic differences through high-content imaging

• Methodological validation standards:

  • Confirm antibody specificity through knockout controls

  • Validate RNAi efficiency with multiple siRNAs or shRNAs targeting different regions

  • Implement rescue experiments with RNAi-resistant constructs

• Integration of seemingly contradictory findings:

  • CIAO1 may function in distinct pathways that appear contradictory but operate in different contexts

  • The dual role in both iron-sulfur cluster assembly and transcriptional regulation may explain contextual differences

  • Temporal aspects of CIAO1 function may reconcile apparently conflicting observations

A particularly informative approach is examining bidirectional genetic interactions, as demonstrated by the finding that not only can Xpd overexpression suppress CIAO1 RNAi defects, but CIAO1 overexpression can also suppress Xpd RNAi phenotypes, suggesting mutual regulation between these genes .

What are promising approaches for investigating CIAO1's role in disease processes?

CIAO1's involvement in fundamental cellular processes suggests several promising avenues for disease-related research:

• Cancer biology applications:

  • Analyze CIAO1 expression in tumor versus normal tissues using immunohistochemistry (1:50-1:500)

  • Investigate correlation between CIAO1 levels and patient prognosis

  • Explore mechanistic connections to WT1, a known tumor suppressor that interacts with CIAO1

• Iron metabolism disorders:

  • Study CIAO1 function in cellular models of iron overload or deficiency

  • Investigate potential links to anemias associated with iron-sulfur cluster defects

  • Analyze CIAO1 genetic variants in patients with unexplained iron disorders

• Neurodegenerative disease connections:

  • Examine CIAO1 in the context of diseases involving iron accumulation in the brain

  • Investigate potential roles in mitochondrial dysfunction

  • Study interactions with proteins implicated in neurodegeneration

• Development of novel methodologies:

  • Design CIAO1 activity reporters to monitor Fe/S protein assembly in real-time

  • Develop targeted approaches to modulate specific CIAO1 interactions

  • Create conditional expression systems to study acute versus chronic CIAO1 loss

• Therapeutic targeting considerations:

  • Assess the potential of targeting CIAO1 interactions as a therapeutic approach

  • Develop screening systems for modulators of CIAO1 function

  • Evaluate potential off-target effects related to disrupting fundamental Fe/S assembly

Research combining CIAO1 antibody-based detection methods with genetic manipulation and functional assays will be most informative in elucidating disease connections.

How can researchers effectively study the relationship between CIAO1 and iron-sulfur cluster targeting proteins?

Investigating the functional relationship between CIAO1 and its Fe/S protein targets requires specialized approaches:

• Fe/S protein maturation assays:

  • Measure activity of Fe/S enzymes (e.g., aconitase, xanthine oxidase) after CIAO1 manipulation

  • Use antibodies against both CIAO1 and target Fe/S proteins in co-localization studies

  • Implement spectroscopic methods to detect Fe/S cluster incorporation

• Structural analysis approaches:

  • Utilize proximity-based proteomic methods to identify the CIAO1 interactome

  • Perform mutational analysis of CIAO1's WD repeat domains to identify client binding regions

  • Apply cryo-EM to visualize the CIA complex architecture

• Temporal dynamics investigation:

  • Use live-cell imaging with fluorescently tagged CIAO1 and client proteins

  • Implement pulse-chase experiments to track newly synthesized Fe/S proteins

  • Analyze cell cycle-dependent changes in CIAO1-client interactions

• Systems biology integration:

  • Create predictive models of the CIA machinery based on experimental data

  • Identify hub proteins and key regulatory nodes in the Fe/S assembly network

  • Perform comparative analysis across species to identify evolutionarily conserved mechanisms

The finding that CIAO1 and Xpd function in a shared pathway, with genetic evidence of mutual regulation, provides a framework for studying CIAO1's relationship with specific Fe/S client proteins . Future studies should build on this to create a comprehensive map of CIAO1-dependent Fe/S protein targeting networks.