CIART Antibody

What is CIART and what are its primary biological functions?

CIART (Circadian-associated transcriptional repressor), also known as CHRONO or C1orf51, functions as a transcriptional repressor in the circadian clock regulatory system. It abrogates the interaction between ARNTL/BMAL1 and the transcriptional coactivator CREBBP, thereby repressing the histone acetyl-transferase action of the CLOCK-ARNTL/BMAL1 heterodimer . This action reduces histone acetylation of target genes. CIART rhythmically binds to E-box components (5'-CACGTG-3') on circadian gene promoters, with its occupancy showing circadian oscillation antiphasic to ARNTL/BMAL1 . More recently, CIART has been identified as a key factor in SARS-CoV-2 infection across multiple tissue types, making it an important target for COVID-19 research .

What validated applications exist for CIART antibodies in research?

Current research validates CIART antibodies for several key applications:

These applications have been validated through extensive testing on tissues and cell lines known to express CIART . When selecting antibodies for research, it is crucial to verify specific reactivity for your experimental model organism, as not all antibodies cross-react across species.

How is CIART expression regulated in different tissues?

CIART expression demonstrates tissue-specific patterns with nuclear localization. It co-localizes with the CLOCK-ARNTL/BMAL1 heterodimer in PML (Promyelocytic Leukemia) bodies within the nucleus . Research indicates that CIART expression follows circadian patterns, with its binding to target promoters occurring in patterns antiphasic to the CLOCK-ARNTL/BMAL1 complex . In studies examining SARS-CoV-2 infection, CIART has been found to be expressed in lung airway organoids, lung alveolar organoids, and cardiomyocytes derived from human pluripotent stem cells, suggesting broad expression across respiratory and cardiovascular tissues .

How does CIART influence SARS-CoV-2 infection, and what antibody-based techniques best demonstrate this relationship?

Recent research using multi-organoid platforms has identified CIART as a critical host factor in SARS-CoV-2 infection . Lung airway organoids, lung alveolar organoids, and cardiomyocytes derived from isogenic CIART−/− human pluripotent stem cells demonstrate significant resistance to SARS-CoV-2 infection, independent of viral entry mechanisms .

For investigating this relationship, recommended antibody-based techniques include:

• Immunohistochemistry (IHC) on infected versus non-infected tissue organoids to visualize CIART expression patterns

• Western blot (WB) analysis to quantify CIART expression levels before and after infection

• Co-immunoprecipitation to identify protein-protein interactions between CIART and viral components

• Immunofluorescence combined with confocal microscopy to observe subcellular localization changes of CIART during infection

Single-cell RNA-sequencing analysis has validated decreased levels of SARS-CoV-2 infection in ciliated-like cells of lung airway organoids lacking CIART , suggesting cell-type specific roles that can be further investigated using antibody-based cell sorting followed by functional assays.

What are the optimal experimental conditions for detecting CIART protein using Western blot techniques?

For optimal Western blot detection of CIART (approximate molecular weight: 41.443 kDa), researchers should consider the following protocol optimizations:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors for cell lysis

  • For nuclear proteins like CIART, include a nuclear extraction step

  • Sonicate briefly to shear genomic DNA and reduce sample viscosity

• Gel electrophoresis conditions:

  • Use 10-12% polyacrylamide gels for optimal resolution of CIART

  • Load 20-40 μg of total protein per lane

• Transfer and blotting:

  • PVDF membranes typically yield better results than nitrocellulose for CIART detection

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary CIART antibody at 1:1000 dilution overnight at 4°C

  • Use HRP-conjugated secondary antibodies at 1:5000 dilution

• Controls:

  • Include positive controls from tissues known to express CIART (brain tissue samples)

  • Use CIART knockout or knockdown samples as negative controls

  • Include housekeeping proteins (β-actin, GAPDH) for normalization

Optimizing these conditions will increase specificity and reduce background, which is particularly important when detecting CIART in complex tissue samples .

How can researchers validate antibody specificity for CIART in experimental settings?

Validating antibody specificity for CIART requires a multi-faceted approach:

• Genetic validation:

  • Compare antibody staining between wild-type and CIART knockout models

  • Use siRNA or shRNA knockdown of CIART followed by Western blot analysis

  • Perform antibody staining in tissues with known differential expression of CIART

• Peptide competition assays:

  • Pre-incubate antibody with excess CIART antigenic peptide

  • Compare staining patterns with and without peptide competition

  • Loss of signal in peptide-competed samples confirms specificity

• Cross-validation with multiple antibodies:

  • Use multiple antibodies targeting different epitopes of CIART

  • Consistent staining patterns across antibodies suggest specificity

• Orthogonal validation:

  • Correlate protein detection with mRNA expression data

  • Confirm subcellular localization matches known distribution (nuclear and PML bodies)

• Mass spectrometry confirmation:

  • Perform immunoprecipitation with the CIART antibody

  • Analyze pulled-down proteins with mass spectrometry to confirm target identity

Researchers should document these validation steps thoroughly, as they significantly strengthen the reliability of experimental findings .

What immunohistochemistry protocols yield optimal results for CIART detection in tissue samples?

For optimal CIART detection in tissue samples via immunohistochemistry, the following protocol adjustments are recommended:

• Fixation and processing:

  • Use 10% neutral buffered formalin for 24-48 hours

  • For nuclear antigens like CIART, avoid overfixation

  • Process tissues into paraffin blocks following standard protocols

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

  • Alternative: EDTA buffer (pH 9.0) if citrate buffer yields insufficient results

  • Allow slides to cool gradually to room temperature (approximately 20 minutes)

• Blocking and antibody incubation:

  • Block endogenous peroxidases with 3% hydrogen peroxide (10 minutes)

  • Block non-specific binding with 5% normal serum from secondary antibody host species

  • Incubate with primary anti-CIART antibody at 1:100-1:200 dilution overnight at 4°C

  • Use highly sensitive detection systems (e.g., polymer-based detection)

• Controls and counterstaining:

  • Include positive control tissues with known CIART expression

  • Use isotype controls to assess non-specific binding

  • Counterstain with hematoxylin to provide cellular context

  • Consider nuclear counterstains to facilitate visualization of CIART's nuclear localization

For multi-label immunofluorescence studies, sequential staining protocols may be necessary to avoid cross-reactivity when examining CIART co-localization with circadian rhythm proteins .

How can researchers effectively use CIART antibodies in organoid-based SARS-CoV-2 infection models?

For effective application of CIART antibodies in organoid-based SARS-CoV-2 infection models, researchers should consider:

• Sample preparation for organoids:

  • Fix organoids in 4% paraformaldehyde for 30-60 minutes

  • For whole-mount staining, permeabilize with 0.5% Triton X-100 for 30 minutes

  • For sectioning, embed in paraffin or OCT compound after fixation

• Antibody optimization for 3D cultures:

  • Increase primary antibody incubation time (24-48 hours) for better penetration

  • Use higher antibody concentrations than for 2D cultures (typically 1.5-2× higher)

  • Include 0.1% Triton X-100 in antibody diluent to improve penetration

• Imaging considerations:

  • Use confocal microscopy for precise localization in 3D structures

  • Consider tissue clearing techniques for whole-mount imaging of larger organoids

  • Acquire z-stack images to capture CIART expression throughout the organoid

• Experimental design for infection studies:

  • Compare CIART expression in mock-infected versus SARS-CoV-2 infected organoids

  • Examine time-course changes in CIART expression following infection

  • Co-stain with viral markers to assess correlation between CIART levels and viral load

• Quantification approaches:

  • Use digital image analysis for quantifying nuclear CIART expression

  • Consider single-cell analysis approaches to account for heterogeneity within organoids

  • Correlate immunostaining results with transcriptomic data when available

These methodological adaptations will help overcome the challenges inherent in working with 3D organoid cultures while generating reliable data on CIART's role in SARS-CoV-2 infection .

What troubleshooting strategies should be employed when CIART antibodies show inconsistent results across experiments?

When facing inconsistent results with CIART antibodies, researchers should systematically troubleshoot using the following strategies:

• Antibody-related factors:

  • Check antibody storage conditions and avoid repeated freeze-thaw cycles

  • Validate antibody lot-to-lot consistency with positive control samples

  • Consider using alternative antibodies targeting different CIART epitopes

  • Prepare fresh working dilutions for each experiment

• Sample-related factors:

  • Ensure consistent sample collection and processing protocols

  • For circadian proteins like CIART, document and standardize sample collection timing

  • Verify protein integrity through total protein stains on membranes

  • Use fresh samples when possible, as CIART may degrade during storage

• Protocol optimization:

  • Systematically test different fixation methods and durations

  • Optimize antigen retrieval conditions (time, temperature, buffer composition)

  • Adjust blocking reagents to reduce background without affecting specific signals

  • Test multiple antibody concentrations and incubation times

• Technical controls:

  • Include both positive and negative controls in every experiment

  • Use internal controls (housekeeping proteins) to normalize for loading variations

  • Perform parallel experiments with validated antibodies to other circadian proteins

  • Document all experimental conditions meticulously for troubleshooting

• Biological variables:

  • Account for circadian expression patterns by standardizing collection times

  • Consider the potential impact of cell cycle phase on nuclear protein expression

  • For disease models, assess how pathological conditions affect antibody accessibility

  • Document donor-to-donor variability in human samples

By systematically addressing these factors, researchers can identify sources of variability and establish reliable protocols for CIART detection across experimental systems .

How can CIART antibodies be utilized in drug discovery efforts targeting SARS-CoV-2 infection?

Given CIART's newly discovered role in SARS-CoV-2 infection, antibodies against this protein can be valuable tools in drug discovery:

• High-throughput screening applications:

  • Develop cell-based assays using CIART antibodies to screen compound libraries

  • Use immunofluorescence-based readouts to quantify CIART expression or localization changes

  • Establish ELISA-based screening methods to identify compounds that modulate CIART-protein interactions

• Target validation approaches:

  • Employ CIART antibodies in ChIP-seq experiments to identify genomic binding sites

  • Use co-immunoprecipitation with CIART antibodies to validate protein-protein interaction disruption by candidate drugs

  • Develop proximity ligation assays to detect changes in CIART interactions following compound treatment

• Mechanism of action studies:

  • Track CIART subcellular localization changes in response to candidate therapeutics

  • Monitor post-translational modifications of CIART using modification-specific antibodies

  • Quantify CIART-dependent pathways in the presence of inhibitory compounds

• Translational research applications:

  • Develop immunohistochemistry panels including CIART for patient sample analysis

  • Correlate CIART expression levels with disease severity or drug responsiveness

  • Establish predictive biomarkers based on CIART expression patterns

Since CIART's effect on SARS-CoV-2 infection has been linked to fatty acid synthesis stimulation , researchers can develop assays combining CIART antibodies with fatty acid metabolism readouts to identify interventions targeting this specific pathway.

What are the considerations for developing mimetic antibodies targeting SARS-CoV-2 based on CIART research?

The development of mimetic antibodies (MAs) targeting SARS-CoV-2 based on CIART research involves several specialized considerations:

• Computational design strategies:

  • Utilize RosettaAntibodyDesign (RAbD) framework for designing antibodies targeting CIART-viral protein interfaces

  • Employ structural bioinformatics approaches to identify critical binding epitopes

  • Calculate design risk ratio (DRR) and antigen risk ratio (ARR) to optimize computational designs

• Scaffold selection considerations:

  • Consider GB1 domain as a structural scaffold for mimetic antibody design

  • Evaluate alternative scaffolds based on stability, expression yield, and binding surface compatibility

  • Balance scaffold size with tissue penetration requirements

• Experimental validation:

  • Validate binding affinity using surface plasmon resonance (SPR) or bio-layer interferometry (BLI)

  • Confirm interaction specificity through competitive binding assays

  • Assess functional activity in cell-based infection models

• Optimization strategies:

  • Iteratively optimize interface residues to enhance binding affinity

  • Improve scaffold stability through consensus design approaches

  • Consider glycosylation engineering to enhance pharmacokinetic properties

• Production and characterization:

  • Establish reproducible expression systems (bacterial, mammalian, or cell-free)

  • Develop purification strategies yielding homogeneous protein preparations

  • Perform thorough biophysical characterization (thermal stability, aggregation propensity)

Researchers should consider combining insights from CIART-dependent SARS-CoV-2 mechanisms with established mimetic antibody design principles to develop novel therapeutic candidates that disrupt this infection pathway .