CIPC Antibody

What is CIPC Antibody and what is its target protein?

CIPC Antibody (such as the PAC041582) is a polyclonal antibody raised in rabbits that targets the CLOCK-interacting pacemaker protein encoded by the CIPC gene. This protein functions as a transcriptional repressor in the circadian clock mechanism by acting as a negative-feedback regulator of CLOCK-ARNTL/BMAL1 transcriptional activity . The antibody allows researchers to detect and analyze the presence, distribution, and function of CIPC protein in various experimental contexts.

The target protein has the following characteristics:

The CIPC protein interacts with CLOCK and forms a ternary complex with the CLOCK-ARNTL/BMAL1 heterodimer, thereby regulating circadian rhythms at the molecular level .

How does CIPC Antibody differ from other circadian rhythm-related antibodies?

CIPC Antibody specifically targets the CLOCK-interacting pacemaker protein, distinguishing it from antibodies that target other components of the circadian machinery such as CLOCK, BMAL1, PER, or CRY proteins. While these other antibodies detect core components of the positive and negative feedback loops of the circadian oscillator, CIPC Antibody recognizes a modulator that fine-tunes the activity of the CLOCK-BMAL1 complex .

This specificity makes CIPC Antibody particularly valuable for investigating the regulatory mechanisms that adjust circadian oscillations. When designing experiments involving multiple circadian proteins, researchers should consider using complementary antibodies with compatible host species to enable co-detection and co-localization studies .

What experimental applications has CIPC Antibody been validated for?

According to available data, CIPC Antibody has been validated for several key experimental applications:

The antibody demonstrates reactivity with both human and mouse samples, making it suitable for comparative studies across these species . When utilizing this antibody for these applications, researchers should carefully consider the recommended dilutions to optimize signal-to-noise ratios.

What are the optimal storage and handling conditions for CIPC Antibody?

For maintaining optimal activity of CIPC Antibody, adhere to these storage and handling guidelines:

• Store the antibody in its preservative buffer (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) at -20°C for long-term storage .

• Aliquot the antibody upon receipt to minimize freeze-thaw cycles, as repeated freezing and thawing can reduce antibody activity.

• When handling the antibody, maintain cold chain conditions using an ice bucket.

• Prior to use, centrifuge the antibody vial briefly to collect the liquid at the bottom.

• For dilutions, use freshly prepared buffers appropriate for the intended application.

Proper storage and handling significantly impact experimental reproducibility. Researchers should maintain detailed records of antibody lot numbers, receipt dates, and freeze-thaw cycles to track potential variations in performance across experiments .

How should I optimize Western blot protocols specifically for CIPC Antibody?

To achieve optimal results with CIPC Antibody in Western blot applications:

• Sample preparation:

  • Use RIPA or NP-40 buffer supplemented with protease inhibitors

  • Load 20-40 μg of total protein per lane

  • Include both positive controls (A431 or NIH/3T3 cell lysates) and negative controls

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels to optimally resolve the 43 kDa CIPC protein

  • Transfer to PVDF membranes at 100V for 1-1.5 hours in cold transfer buffer

• Immunoblotting:

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

  • Incubate with primary CIPC Antibody at 1:1000-1:5000 dilution overnight at 4°C

  • Wash 3-5 times with TBST

  • Incubate with HRP-conjugated goat anti-rabbit secondary antibody (1:10000) for 1 hour

  • Develop using enhanced chemiluminescence substrate

• Expected results:

  • The target band should appear at approximately 43 kDa

  • Validate specificity by comparing with predicted molecular weight and positive controls

For circadian rhythm studies, consider harvesting cells at different time points across a 24-hour cycle to capture temporal variations in CIPC protein expression.

What are the critical considerations for immunohistochemistry experiments using CIPC Antibody?

For successful immunohistochemistry with CIPC Antibody:

• Tissue preparation:

  • Use 4% paraformaldehyde-fixed, paraffin-embedded sections (4-6 μm thick)

  • Include both normal and disease tissues (e.g., human stomach and liver cancer) for comparison

• Antigen retrieval:

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Boil sections for 15-20 minutes followed by cooling to room temperature

• Immunostaining protocol:

  • Block endogenous peroxidase with 3% H₂O₂

  • Apply protein block (5% normal goat serum)

  • Incubate with CIPC Antibody (1:20-1:200 dilution) overnight at 4°C

  • Use HRP-conjugated secondary antibody and DAB substrate for detection

  • Counterstain with hematoxylin

• Controls and validation:

  • Include negative controls (omitting primary antibody)

  • Validate nuclear localization pattern consistent with CIPC's function

  • Compare staining patterns in normal versus diseased tissues

When interpreting IHC results, note that CIPC expression may vary with circadian timing, so documenting the time of tissue collection is advisable for circadian studies .

How can I use CIPC Antibody to investigate circadian rhythm disruptions in disease models?

To employ CIPC Antibody for studying circadian disruptions in disease contexts:

• Experimental design considerations:

  • Establish a time-course sampling protocol (every 4 hours across 24-48 hours)

  • Compare CIPC expression and localization between healthy and disease models

  • Synchronize cells prior to analysis (serum shock or dexamethasone treatment)

• Multi-method approach:

  • Use Western blot to quantify total CIPC protein levels across time points

  • Employ immunofluorescence to track subcellular localization changes

  • Complement protein data with CIPC mRNA expression analysis (qPCR)

• Functional analysis:

  • Combine with co-immunoprecipitation to assess CIPC-CLOCK interactions

  • Investigate CIPC phosphorylation states using phospho-specific antibodies

  • Correlate CIPC expression patterns with circadian output genes

This approach has revealed altered CIPC expression and localization patterns in metabolic disorders, cancer, and neurodegenerative diseases, providing insights into how circadian disruption contributes to pathogenesis .

For cancer research specifically, comparing CIPC expression between tumor and adjacent normal tissues can reveal associations between circadian dysregulation and malignant transformation .

What approaches can resolve contradictory results when using CIPC Antibody across different experimental systems?

When facing contradictory results with CIPC Antibody:

• Systematic validation:

  • Confirm antibody specificity using knockout/knockdown controls

  • Test multiple antibody lots and sources if available

  • Validate with orthogonal detection methods (mass spectrometry)

• Technical troubleshooting:

  • Optimize protein extraction methods for different sample types

  • Adjust antibody concentration and incubation conditions

  • Evaluate potential interfering factors (detergents, fixatives)

• Biological considerations:

  • Account for circadian timing differences between experiments

  • Consider species-specific variations in CIPC structure and expression

  • Assess potential post-translational modifications affecting epitope recognition

• Data interpretation:

  • Document complete experimental conditions and timing

  • Consider contextual differences between in vitro and in vivo systems

  • Analyze whether contradictions reflect biological complexity rather than technical issues

One common source of apparent contradictions is the time-dependent nature of CIPC expression. Standardizing the time of sample collection across experiments is crucial for meaningful comparisons .

How can I combine CIPC Antibody with other molecular tools to comprehensively study circadian transcriptional regulation?

A multi-faceted approach to studying circadian transcriptional regulation:

• Chromatin immunoprecipitation (ChIP) strategies:

  • Use CIPC Antibody for ChIP to identify genomic binding sites

  • Perform sequential ChIP with CLOCK and BMAL1 antibodies to identify co-occupied regions

  • Integrate with RNA-Seq to correlate binding with transcriptional outcomes

• Protein-protein interaction studies:

  • Employ co-immunoprecipitation with CIPC Antibody to pull down interaction partners

  • Use proximity ligation assays to visualize CIPC-CLOCK interactions in situ

  • Complement with live-cell imaging using fluorescently tagged proteins

• Functional genomics integration:

  • Combine with CRISPR-Cas9 editing of CIPC to assess functional consequences

  • Correlate with rhythmic transcriptome and proteome datasets

  • Develop mathematical models incorporating CIPC regulatory dynamics

This integrated approach provides mechanistic insights into how CIPC modulates circadian transcription by stimulating ARNTL/BMAL1-dependent phosphorylation of CLOCK and repressing CLOCK-ARNTL/BMAL1 transcriptional activity .

What are the most common causes of false-positive and false-negative results when using CIPC Antibody?

Understanding and addressing potential sources of error:

False-positive results:

• Non-specific binding due to excessive antibody concentration or insufficient blocking

• Cross-reactivity with structurally similar proteins

• Endogenous peroxidase activity in tissue samples not properly quenched

• Sample contamination during processing

• Secondary antibody binding in the absence of primary antibody

False-negative results:

• Epitope masking due to improper fixation or inadequate antigen retrieval

• Protein degradation during sample preparation

• Insufficient antibody concentration or incubation time

• Sub-optimal detection system sensitivity

• Time-dependent expression fluctuations (CIPC levels vary throughout circadian cycle)

Mitigation strategies:

• Always include positive and negative controls

• Validate with multiple detection methods when possible

• For circadian proteins like CIPC, document sampling time and synchronization status

• Consider performing pilot experiments with a dilution series to determine optimal antibody concentration

• When troubleshooting, change only one variable at a time

How do I quantitatively analyze CIPC expression patterns across different circadian time points?

For rigorous quantitative analysis of CIPC expression across the circadian cycle:

• Experimental design:

  • Sample at minimum 6 time points across 24 hours (ideally every 4 hours)

  • Include at least 3 biological replicates per time point

  • Synchronize cells/tissues before beginning time course (serum shock, dexamethasone, or light-dark cycles)

• Western blot quantification:

  • Use housekeeping proteins unaffected by circadian rhythms for normalization

  • Apply densitometry software with appropriate background subtraction

  • Present data as relative expression normalized to peak expression or time-point zero

• Image analysis for immunofluorescence/IHC:

  • Quantify nuclear vs. cytoplasmic signal intensities

  • Measure percentage of CIPC-positive cells across time points

  • Track changes in subcellular localization

• Statistical analysis:

  • Apply cosinor analysis to test for rhythmicity

  • Determine amplitude, period, and phase of oscillations

  • Use appropriate statistical tests to compare parameters between experimental groups

• Data presentation:

  • Plot expression values against circadian/zeitgeber time

  • Include error bars representing standard error or deviation

  • Fit rhythmic data with cosine curves when appropriate

This approach allows for precise characterization of how experimental manipulations affect CIPC's circadian expression pattern and subsequent effects on cellular rhythmicity .

What critical controls should be included when validating CIPC Antibody for new experimental systems?

Comprehensive validation requires these essential controls:

• Positive controls:

  • Known CIPC-expressing cell lines (A431, NIH/3T3)

  • Tissues with documented CIPC expression (human stomach, liver)

  • Recombinant CIPC protein as Western blot standard

• Negative controls:

  • CIPC knockout/knockdown samples

  • Secondary antibody-only controls

  • Isotype controls (irrelevant antibody from same species/isotype)

• Specificity controls:

  • Peptide competition assay (pre-incubation with immunizing peptide)

  • Detection of expected molecular weight band (43 kDa)

  • Correlation with mRNA expression data

• Application-specific controls:

  • For Western blots: loading controls and molecular weight markers

  • For IHC/IF: tissue controls with known expression patterns

  • For circadian studies: time-course controls with known rhythmic proteins

• Cross-validation:

  • Comparison with alternative CIPC antibodies (different clones/epitopes)

  • Correlation with orthogonal detection methods

  • Multi-species validation if working with non-human models

Thorough validation not only ensures reliable results but also helps identify optimal experimental conditions for each new system or application .

How can CIPC Antibody be used in studies investigating the interface between circadian rhythms and cellular metabolism?

CIPC Antibody enables investigation of the circadian-metabolic interface through:

• Co-localization studies:

  • Dual immunofluorescence with CIPC Antibody and metabolic sensors

  • Tracking CIPC localization in response to metabolic perturbations

  • Visualizing interactions with metabolic transcription factors

• Temporal coordination analysis:

  • Monitoring CIPC expression/activity alongside metabolic oscillations

  • Correlating CIPC dynamics with fluctuations in cellular energy status

  • Investigating phase relationships between CIPC and metabolic rhythms

• Mechanistic investigations:

  • Examining how CIPC mediates cross-talk between circadian and metabolic pathways

  • Determining how metabolic signals modulate CIPC's repressive function

  • Investigating CIPC's role in synchronizing peripheral clocks to feeding cycles

• Disease model applications:

  • Analyzing CIPC dysregulation in metabolic disorders

  • Investigating therapeutic interventions targeting CIPC-mediated pathways

  • Studying how time-restricted feeding impacts CIPC function

This research direction has significant implications for understanding metabolic diseases with circadian components, including diabetes, obesity, and non-alcoholic fatty liver disease .

What methodological considerations apply when using CIPC Antibody in multiplexed imaging approaches?

For successful multiplexed imaging with CIPC Antibody:

• Antibody compatibility planning:

  • Select primary antibodies from different host species

  • When using multiple rabbit antibodies, employ sequential immunostaining with stripping steps

  • Consider using directly conjugated CIPC Antibody for one channel

• Signal separation strategies:

  • Use fluorophores with minimal spectral overlap

  • Apply linear unmixing algorithms for closely adjacent emissions

  • Employ sequential scanning for confocal microscopy

• Validation for multiplexed applications:

  • Test antibodies individually before combining

  • Include single-stained controls for spectral overlap assessment

  • Verify that multiplexing doesn't alter individual staining patterns

• Sample preparation optimization:

  • Select fixation methods compatible with all target epitopes

  • Optimize antigen retrieval conditions for multiple targets

  • Adjust blocking to prevent cross-reactivity in multiplexed settings

• Analysis considerations:

  • Employ computational methods to quantify co-localization

  • Use machine learning approaches for pattern recognition

  • Apply spatial statistics to characterize protein distribution relationships

Multiplexed imaging with CIPC Antibody is particularly valuable for simultaneously visualizing interactions between clock proteins and their regulatory partners or downstream targets .