CYC1-2 Antibody

What is CYC1 and why is it a target for antibody-based detection in research?

CYC1 (Cytochrome c-1) is a critical subunit of the cytochrome bc1 complex (complex III) in the mitochondrial electron transport chain. It functions as a key respiratory component among the 11 subunits of this complex, mediating electron transfer from the Rieske iron-sulfur protein to cytochrome c. This process is essential for cellular respiration and energy production in the form of ATP .

CYC1 is particularly important in research because dysregulation of this protein has been linked to metabolic disorders, neurodegenerative diseases, and cancer, making it a promising target for therapeutic interventions . Understanding CYC1's role in cellular energy production is crucial for developing strategies to treat conditions associated with mitochondrial dysfunction. The protein is localized to the mitochondrial inner membrane, with specific intermembrane side positioning and single-pass membrane topology . Its calculated molecular weight is approximately 35 kDa, which matches observed experimental values in Western blot applications .

What are the validated applications and species reactivity for commercially available CYC1 antibodies?

Commercial CYC1 antibodies have been validated for several key applications with specific reactivity patterns:

Species reactivity has been confirmed for:

• Human (primary validation)

• Mouse (primary validation)

• Rat (cited reactivity)

• Pig (cited reactivity)

• Monkey (cited reactivity)

Positive reactivity has been specifically demonstrated in human brain tissue (WB) and human cancer tissues including liver cancer and breast cancer (IHC) . For mouse samples, successful detection has been reported in multiple tissues including brain, kidney, liver, and heart .

How do polyclonal and monoclonal CYC1 antibodies differ in their research applications?

Polyclonal CYC1 antibodies, such as the rabbit polyclonal antibody (10242-1-AP), recognize multiple epitopes on the CYC1 protein, offering advantages in detection sensitivity but potentially lower specificity compared to monoclonal alternatives .

Polyclonal antibodies are particularly useful when:

• Signal amplification is needed for low-abundance targets

• Detecting denatured proteins in applications like Western blot

• The native protein conformation is unknown or variable

• Cross-species reactivity is desired (as evidenced by the broad reactivity of available polyclonal CYC1 antibodies)

For instance, the polyclonal antibody CAB10449 specifically targets a sequence corresponding to amino acids 85-291 of human CYC1 (NP_001907.2), which includes critical functional domains . This broad epitope recognition makes it suitable for detecting CYC1 across multiple experimental conditions.

When higher specificity is required, particularly for distinguishing between closely related cytochrome family members or for therapeutic applications, monoclonal antibodies would be preferred, though specific information about monoclonal CYC1 antibodies was not provided in the available literature.

What protocol modifications are recommended when using CYC1 antibodies for immunohistochemistry of fixed tissues?

When performing immunohistochemistry (IHC) with CYC1 antibodies on fixed tissues, several protocol optimizations are recommended:

• Antigen retrieval: The preferred method is TE buffer at pH 9.0, although citrate buffer at pH 6.0 can serve as an alternative . This step is critical because formaldehyde fixation can mask epitopes through protein cross-linking.

• Antibody dilution: For optimal results in IHC applications, CYC1 antibodies should be used at dilutions ranging from 1:50 to 1:500 . Sample-dependent titration is strongly recommended to determine optimal concentration for each tissue type.

• Fixation considerations: Studies have shown that fixation protocols significantly impact antibody epitope recognition. In comprehensive antibody screening experiments, samples are typically processed with both fixed (1.6% formaldehyde in PBS for 20 minutes) and unfixed conditions to evaluate epitope sensitivity to fixation .

• Tissue-specific validation: Particularly strong positive staining has been reported in human liver cancer tissue and breast cancer tissue , making these suitable positive controls for protocol optimization.

• Blocking optimization: Due to the mitochondrial localization of CYC1, tissues with high mitochondrial content may produce higher background. Extended blocking (5% BSA or serum from the secondary antibody species) for 1-2 hours at room temperature is advised to minimize non-specific binding.

What are the best practices for Western blot detection of CYC1 protein?

For optimal Western blot detection of CYC1 protein:

How can researchers validate CYC1 antibody specificity using genetic approaches?

Validating CYC1 antibody specificity using genetic approaches is essential for ensuring reliable experimental results:

• Knockout/knockdown validation: The literature indicates successful use of CYC1 antibodies in knockout/knockdown studies . Researchers should:

  • Generate CRISPR/Cas9 knockouts or siRNA knockdowns of CYC1

  • Perform parallel Western blot analysis of wild-type and knockout/knockdown samples

  • Confirm specific band disappearance or reduction in knockout/knockdown samples

• Overexpression systems: Complementary to knockout approaches, overexpression of tagged CYC1 constructs can be used to:

  • Confirm band position shift with fusion tags

  • Demonstrate increased signal intensity correlating with expression level

  • Validate subcellular localization through co-staining experiments

• Peptide competition: Pre-incubating the antibody with the immunizing peptide (the recombinant fusion protein containing amino acids 85-291 of human CYC1) should eliminate specific staining in Western blot and IHC applications.

• Cross-species validation: The reported cross-reactivity of CYC1 antibodies with human, mouse, and rat samples can be leveraged to confirm consistent detection patterns across species, accounting for evolutionary conservation of the protein.

• Genetic variant analysis: Similar to approaches used with CYP enzyme variants , researchers can examine how genetic polymorphisms in CYC1 might affect antibody binding, particularly in human samples with known genetic variations.

How can CYC1 antibodies be utilized in studies of mitochondrial dysfunction and metabolic disorders?

CYC1 antibodies serve as valuable tools for investigating mitochondrial dysfunction and metabolic disorders through several advanced approaches:

• Bioenergetic profiling: CYC1, as a critical component of the electron transport chain, can be quantified relative to other respiratory chain components to assess mitochondrial integrity. Researchers can:

  • Perform immunoblotting of CYC1 alongside other complex III subunits to evaluate stoichiometric relationships

  • Correlate CYC1 expression levels with oxygen consumption rate measurements

  • Assess changes in CYC1 levels in response to metabolic stressors or drug treatments

• Disease model characterization: CYC1 antibodies can be used to study pathological conditions:

  • Neurodegenerative diseases often feature mitochondrial dysfunction

  • Cancer cells frequently exhibit altered energy metabolism (Warburg effect)

  • Metabolic disorders may show aberrant expression or post-translational modifications of CYC1

• Tissue-specific mitochondrial adaptations: By comparing CYC1 expression across tissues with varying metabolic demands:

  • High-energy tissues (brain, heart, liver) versus low-energy tissues

  • Adaptive responses to exercise, caloric restriction, or hypoxia

  • Developmental changes in mitochondrial content and composition

• Pharmacological intervention assessment: CYC1 antibodies can monitor mitochondrial responses to:

  • Mitochondrially-targeted drugs

  • Toxicants affecting respiratory chain function

  • Therapeutic candidates for mitochondrial disorders

What considerations are important when designing co-immunoprecipitation experiments using CYC1 antibodies?

When designing co-immunoprecipitation (Co-IP) experiments to study CYC1 interactions:

• Membrane protein considerations: As CYC1 is a mitochondrial membrane protein , specialized lysis conditions are required:

  • Digitonin (0.5-1%) or n-dodecyl-β-D-maltoside (0.5-1%) buffers maintain native protein complexes better than harsher detergents

  • Lysis buffers should include protease inhibitors and be performed at 4°C to prevent protein degradation

  • Gentle homogenization techniques help preserve membrane protein complexes

• Antibody orientation: Both approaches have advantages:

  • CYC1 antibody as the pull-down antibody: Directly captures CYC1 and associated proteins

  • Partner protein antibody as the pull-down: Can validate interactions from the opposite perspective

• Controls for specificity:

  • IgG control from the same species as the antibody

  • Immunodepleted or knockout cell lysates

  • Peptide competition to block specific antibody binding

• Interaction validation approaches:

  • Reverse Co-IP (switching antibodies)

  • Proximity ligation assay as an orthogonal method

  • Yeast three-hybrid systems similar to those used for protein-protein interaction studies

• Complex stability considerations:

  • The cytochrome bc1 complex may disassemble under certain buffer conditions

  • Cross-linking prior to lysis can stabilize transient interactions

  • Native gel electrophoresis can be used to verify complex integrity

What approaches can be used to study CYC1 in protein-protein interaction networks?

Multiple methodologies can be employed to investigate CYC1's role in protein-protein interaction networks:

• Yeast hybrid systems: Similar to the yeast three-hybrid assay used for studying protein interactions , researchers can:

  • Express CYC1 fused to a DNA-binding domain

  • Screen for interacting partners using activation domain fusion libraries

  • Validate specific interactions with targeted constructs

  • Use recombinant antibody fragments to selectively inhibit specific interactions in vivo

• Proximity-based approaches:

  • BioID or TurboID fusion with CYC1 for proximity labeling of interaction partners

  • APEX2 technology for spatially-restricted biotinylation of proximal proteins

  • These methods are particularly valuable for membrane proteins like CYC1 where traditional pull-downs may disrupt interactions

• Fluorescence-based interaction studies:

  • Förster Resonance Energy Transfer (FRET) between fluorescently-tagged CYC1 and candidate partners

  • Fluorescence Complementation Assays (BiFC) to visualize interactions in living cells

  • Fluorescence Correlation Spectroscopy (FCS) to assess binding dynamics

• Mass spectrometry-based interactomics:

  • Immunoprecipitation coupled with mass spectrometry for unbiased interaction discovery

  • Cross-linking mass spectrometry (XL-MS) to map interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify conformational changes upon binding

• Recombinant antibody technology: As demonstrated in studies of protein-protein interactions, recombinant antibodies can be developed to:

  • Target specific interaction interfaces

  • Inhibit particular protein-protein interactions in vivo

  • Serve as research tools for functional validation of interactions

What are common issues encountered when using CYC1 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with CYC1 antibodies:

• Non-specific binding: This is particularly problematic in tissues with high mitochondrial content.

  • Solution: Extended blocking (2 hours at room temperature) with 5% BSA or serum matching the secondary antibody species

  • Solution: Titrate primary antibody concentration; try more stringent washing with PBS-T (0.1% Tween-20)

  • Solution: Pre-absorb antibody with liver powder to reduce non-specific interactions

• Inconsistent batch-to-batch performance:

  • Solution: Validate each new lot against previously successful lots

  • Solution: Maintain internal positive controls (e.g., human brain tissue or A-549 cells)

  • Solution: Consider pooling antibody aliquots from verified batches for long-term studies

• Fixation-sensitive epitopes: As demonstrated in comprehensive antibody staining protocols, fixation can significantly alter epitope accessibility .

  • Solution: Compare fixed (1.6% formaldehyde, 20 minutes) versus unfixed samples

  • Solution: Optimize antigen retrieval using both TE buffer (pH 9.0) and citrate buffer (pH 6.0)

  • Solution: Adjust fixation time and fixative concentration

• Mitochondrial isolation challenges:

  • Solution: Use gentle homogenization techniques to preserve mitochondrial integrity

  • Solution: Include protease inhibitors in all buffers to prevent degradation

  • Solution: Consider subcellular fractionation to enrich for mitochondria prior to analysis

• Cross-reactivity with other cytochrome family members:

  • Solution: Confirm specificity using knockout/knockdown approaches

  • Solution: Compare staining patterns with antibodies targeting other cytochrome proteins

  • Solution: Perform peptide competition assays to confirm specificity

How can researchers standardize CYC1 antibody-based assays across different studies?

Standardization is critical for reproducibility and comparative analysis across studies:

• Antibody validation framework:

  • Implement tiered validation approaches similar to those used in comprehensive antibody staining databases

  • Document antibody performance in knockout/knockdown models

  • Register validated antibodies in community databases with experimental protocols

• Reference sample inclusion:

  • Maintain common positive control samples across experiments

  • Include standardized cell lines with known CYC1 expression levels

  • Create standard curves for quantitative applications

• Protocol standardization:

  • Develop detailed standard operating procedures (SOPs) for each application

  • Specify key variables including antibody concentrations, incubation times/temperatures, and buffer compositions

  • Address sample-specific optimization requirements systematically

• Combinatorial barcoding strategies:

  • Implement CD45-based or similar barcoding schemes for multiplexed analysis

  • Pool samples to minimize batch effects and technical variability

  • Process experimental and control samples simultaneously

• Reporting standards:

  • Document complete antibody information (catalog number, lot, dilution, incubation conditions)

  • Include comprehensive methods sections detailing sample preparation and analysis

  • Share raw data and analysis workflows through repositories

What controls should be included when validating CYC1 antibody specificity and sensitivity?

A comprehensive validation strategy requires multiple controls:

• Positive controls:

  • Human brain tissue for Western blot applications

  • Human liver cancer and breast cancer tissues for IHC applications

  • Cell lines with confirmed CYC1 expression: A-549, A375, LO2, 22Rv1

  • Mouse tissues: brain, kidney, liver, heart

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG) at equivalent concentration

  • Pre-absorption with immunizing peptide

  • CRISPR/siRNA knockout/knockdown samples

• Specificity controls:

  • Cross-reactivity assessment with related cytochrome family members

  • Testing across multiple species to confirm evolutionary conservation of epitopes

  • Peptide competition assays using the immunizing sequence (amino acids 85-291 of human CYC1)

• Method-specific controls:

  • For IHC: Adjacent tissue sections with alternative antibody targeting CYC1

  • For WB: Molecular weight markers to confirm band position at 35 kDa

  • For IP: Non-specific IgG pull-down control

  • For fluorescence microscopy: Co-localization with established mitochondrial markers

• Quantification controls:

  • Serial dilution series to establish linear dynamic range

  • Recombinant protein standards for absolute quantification

  • Internal loading controls appropriate for mitochondrial proteins

How are CYC1 antibodies being utilized in studies of autoimmune conditions and drug hypersensitivity?

While CYC1-specific autoantibodies have not been extensively studied, the field of xenobiotic-induced autoimmunity provides valuable parallels:

• Drug-induced autoimmunity models: Similar to studies of trichloroethylene hypersensitivity syndrome (TCE-HS) and anti-CYP2E1 autoantibodies , CYC1 antibodies can be used to:

  • Investigate whether drug-induced mitochondrial damage leads to CYC1 autoantibody production

  • Assess correlation between mitochondrial protein autoantibodies and clinical manifestations

  • Evaluate genetic susceptibility factors (such as HLA polymorphisms) in mitochondrial autoimmunity

• Biomarker development:

  • Quantification of anti-CYC1 autoantibodies in patient sera as potential biomarkers

  • Correlation with disease severity or treatment response

  • Integration with other mitochondrial autoantibody measurements for comprehensive profiles

• Mechanistic studies:

  • Investigation of how mitochondrial stress might lead to CYC1 epitope exposure to the immune system

  • Examination of post-translational modifications that might create neo-epitopes

  • Assessment of cross-reactivity between microbial antigens and mitochondrial proteins (molecular mimicry)

The anti-CYP2E1 autoantibody research provides a model for such studies, where significantly elevated antibody levels were observed in exposed groups compared to controls, suggesting a mechanism for hypersensitivity development .

What role can recombinant antibody technology play in developing CYC1-targeted research tools?

Recombinant antibody technology offers significant advantages for developing next-generation CYC1 research tools:

• Targeted inhibition of protein-protein interactions:

  • Similar to the approach demonstrated for AHP3-CKI1 interactions , recombinant antibodies can be designed to:

  • Target specific interaction interfaces on CYC1

  • Selectively inhibit or enhance particular protein-protein interactions

  • Provide temporal control through inducible expression systems

• Intrabody development:

  • Creation of scFv (single-chain variable fragment) antibodies that function within cells

  • Targeting to specific subcellular compartments through localization signals

  • Development of conformation-specific intrabodies to distinguish active/inactive states

• Functionalized antibody fragments:

  • Fusion with fluorescent proteins for real-time interaction monitoring

  • Integration with degradation-inducing domains for targeted protein knockdown

  • Coupling with enzymatic domains for proximity labeling applications

• High-throughput selection methods:

  • Phage display libraries to generate highly specific CYC1-targeting antibodies

  • Yeast surface display for affinity maturation

  • In vitro evolution approaches to optimize binding properties

• Standardization advantages:

  • Recombinant production ensures batch-to-batch consistency

  • Detailed molecular characterization of binding properties

  • Possibility for distribution of antibody-encoding plasmids rather than protein

As demonstrated in the recombinant antibody work for protein-protein interaction inhibition, such tools can be used effectively in three-hybrid assays and potentially in more complex cellular systems .

How might systems biology approaches integrate CYC1 antibody data with other mitochondrial function metrics?

Integration of CYC1 antibody data into systems biology frameworks offers powerful approaches to understanding mitochondrial biology:

• Multi-omics integration:

  • Correlation of CYC1 protein levels (antibody-based detection) with transcriptomic data

  • Integration with metabolomic profiles of TCA cycle and electron transport chain intermediates

  • Incorporation of proteomic data on post-translational modifications

  • Analysis alongside genomic variation data, particularly for mitochondrial genes

• Network modeling approaches:

  • Positioning CYC1 within protein-protein interaction networks

  • Flux balance analysis incorporating CYC1 abundance data

  • Bayesian network models predicting mitochondrial function from multi-parameter datasets

• High-content screening platforms:

  • Multiplexed antibody-based detection of mitochondrial proteins

  • Integration with functional readouts (membrane potential, ROS production)

  • Correlation with cellular phenotypes in disease models or drug screening

• Single-cell approaches:

  • Mass cytometry (CyTOF) incorporating CYC1 antibodies alongside other markers

  • Development of barcoding strategies similar to those used in antibody screening

  • Single-cell proteomics to assess mitochondrial heterogeneity within populations

• Computational modeling:

  • Predictive models of electron transport chain function incorporating protein abundance data

  • In silico prediction of drug effects on mitochondrial function

  • Machine learning approaches integrating antibody-based measurements with functional outcomes

These integrated approaches can provide systems-level insights into mitochondrial biology that would not be possible through single-parameter analyses alone.