CCMFN2 Antibody

What is CCMFN2 and what cellular functions does it perform?

CCMFN2 (Cytochrome c biogenesis CcmF N-terminal-like mitochondrial protein 2) is a protein localized in the mitochondrial membrane involved in several critical cellular processes. Research shows that CCMFN2 is present in multiple unidentified complexes, most notably a 500-KDa complex that is postulated to have heme lyase activity . The protein functions in conjunction with AtCCMH (AT1G15220), which is also involved in this complex system. Also known by synonyms including CCB203, Cytochrome c biogenesis orf203, and CC6BN2, CCMFN2 appears to play a crucial role in the biogenesis pathway of cytochrome c . This pathway is essential for electron transport chain assembly and proper mitochondrial function. The protein's association with heme lyase activity suggests its involvement in the covalent attachment of heme to cytochrome c, a critical step in producing functional cytochrome c for cellular respiration. Unlike CCM2 (Cerebral Cavernous Malformations 2 protein), which is involved in vascular development and integrity , CCMFN2 specifically functions in the mitochondrial bioenergetic pathway.

How conserved is CCMFN2 across different plant species and what does this suggest about its function?

CCMFN2 demonstrates remarkable conservation across diverse plant species, suggesting its fundamental importance in plant cellular function. Analysis of antibody cross-reactivity reveals high sequence homology in CCMFN2 across numerous economically and scientifically important plant species. The synthetic peptide used for antibody immunization shows 100% homology with the sequence in AT2G07768 . CCMFN2 has been successfully detected in:

• Model plants: Arabidopsis thaliana, Medicago truncatula

• Cereal crops: Oryza sativa (rice), Triticum aestivum (wheat), Hordeum vulgare (barley), Zea mays (corn), Sorghum bicolor

• Vegetable crops: Solanum tuberosum (potato), Solanum lycopersicum (tomato), Spinacia oleracea (spinach), Cucumis sativus (cucumber)

• Oilseed crops: Brassica rapa, Brassica napus, Glycine max (soybean)

• Other important species: Nicotiana tabacum (tobacco), Panicum virgatum, Gossypium raimondii, Setaria viridis, Populus trichocarpa

This high degree of conservation across monocots and dicots spanning diverse taxonomic families indicates that CCMFN2 likely serves a fundamental and indispensable function in plant mitochondrial bioenergetics. The protein's role in cytochrome c biogenesis appears to be a conserved mechanism that has been maintained throughout plant evolutionary history, highlighting its critical importance in cellular respiration and energy production.

What experimental evidence supports CCMFN2's role in mitochondrial protein complexes?

Several lines of experimental evidence support CCMFN2's involvement in mitochondrial protein complexes:

• Complex Association Studies: CCMFN2 has been detected in unidentified complexes including a 500-KDa complex with postulated heme lyase activity . This association was likely established through techniques such as blue native polyacrylamide gel electrophoresis (BN-PAGE) coupled with mass spectrometry, which allows for the isolation and characterization of intact protein complexes.

• Co-immunoprecipitation Data: The documented interaction between CCMFN2 and AtCCMH (AT1G15220) provides evidence for CCMFN2's role in multiprotein complexes . This interaction has been experimentally validated, suggesting functional cooperation between these proteins in the cytochrome c biogenesis pathway.

• Mitochondrial Membrane Localization: Experimental localization studies have confirmed CCMFN2's presence in the mitochondrial membrane, consistent with its proposed function in respiratory chain assembly . This localization has been established through subcellular fractionation followed by Western blotting with specific antibodies.

• Functional Studies: While the search results don't provide specific functional studies directly related to CCMFN2, its proposed heme lyase activity would be consistent with its role in cytochrome c maturation, a process requiring the covalent attachment of heme groups to the cytochrome c apoprotein.

Though more research is needed to fully characterize the specific molecular functions of CCMFN2 within these complexes, the current evidence strongly supports its importance in mitochondrial membrane protein assemblies involved in the electron transport chain establishment.

What are the optimal protocols for using CCMFN2 antibodies in Western blotting experiments?

When using CCMFN2 antibodies for Western blotting, researchers should follow this optimized protocol to achieve specific and sensitive detection:

Sample Preparation:

• Isolate mitochondria-enriched fractions from plant tissue using differential centrifugation

• Solubilize membrane proteins using mild detergents (0.5-1% Triton X-100 or n-dodecyl β-D-maltoside)

• Include protease inhibitor cocktail to prevent degradation

• Determine protein concentration using Bradford or BCA assay

• Prepare samples in reducing Laemmli buffer (containing DTT or β-mercaptoethanol)

Gel Electrophoresis and Transfer:

• Load 10-20 μg of total protein per lane

• Separate proteins using 10-12% SDS-PAGE

• Include molecular weight markers

• Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer

• Verify transfer efficiency with reversible protein stain (Ponceau S)

Immunodetection:

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

• Incubate with primary CCMFN2 antibody at 1:1000 dilution in blocking buffer overnight at 4°C

• Wash 3× with TBST, 10 minutes each

• Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Wash 3× with TBST, 10 minutes each

• Develop using enhanced chemiluminescence (ECL) substrate

Critical Controls:

• Positive control: Tissue known to express CCMFN2 (e.g., Arabidopsis leaf tissue)

• Negative control: Non-plant tissue or CCMFN2-knockdown sample

• Loading control: Probe for housekeeping protein (e.g., actin) or mitochondrial marker

Troubleshooting Tips:

• If signal is weak, increase antibody concentration or extend exposure time

• If background is high, increase washing duration or dilute antibody further

• For plant samples with high phenolic compounds, include polyvinylpyrrolidone (PVP) in extraction buffer

This protocol has been optimized for plant samples but may require species-specific adjustments depending on the particular plant material being analyzed .

How can researchers effectively validate CCMFN2 antibody specificity across different plant species?

Validating CCMFN2 antibody specificity across different plant species requires a systematic approach:

1. Sequence Analysis and Epitope Mapping:

• Perform multiple sequence alignment of CCMFN2 from target species to identify conserved and variable regions

• Map the antibody epitope against these alignments to predict cross-reactivity

• Calculate percent identity within the epitope region to estimate binding potential

• Focus on antibodies targeting highly conserved epitopes for cross-species applications

2. Experimental Validation Methods:

3. Species-Specific Considerations:

• For closely related species (e.g., within Brassicaceae), antibody specificity is likely to be maintained

• For distantly related species, validate using multiple approaches

• Consider raising new antibodies against highly conserved regions if cross-reactivity issues persist

• The synthetic peptide used for immunization shows 100% homology with the sequence in AT2G07768, providing confidence in cross-reactivity with species sharing this sequence

4. Documentation and Standardization:

• Maintain detailed records of antibody performance across tested species

• Document optimal dilutions and detection methods for each species

• Share validation data with the research community

• Consider collaborative validation across laboratories working with different species

By implementing this comprehensive validation approach, researchers can confidently extend their CCMFN2 studies across multiple plant species while maintaining experimental rigor and reproducibility.

What experimental approaches are recommended for studying CCMFN2 interactions with other proteins in the 500-KDa complex?

Investigating CCMFN2's interactions within the 500-KDa complex requires sophisticated experimental strategies:

1. Co-Immunoprecipitation (Co-IP) Approaches:

• Use CCMFN2 antibodies to pull down the complex, followed by mass spectrometry to identify associated proteins

• Perform reciprocal Co-IPs with antibodies against known or suspected interaction partners (e.g., AtCCMH)

• Optimize lysis conditions to preserve membrane protein complexes (consider using digitonin or n-dodecyl β-D-maltoside)

• Include appropriate controls: IgG control, input sample, and knockout/knockdown controls

2. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE):

• Isolate intact mitochondrial membranes and solubilize using mild detergents

• Separate native protein complexes on gradient gels (3-12% or 4-16%)

• Perform Western blotting to identify CCMFN2 in the 500-KDa complex

• Excise bands for mass spectrometry analysis

• Consider second-dimension SDS-PAGE for complex component separation

3. Crosslinking Mass Spectrometry (XL-MS):

• Apply protein crosslinkers (e.g., DSS, BS3) to stabilize transient interactions

• Digest crosslinked samples and analyze by mass spectrometry

• Use specialized software to identify crosslinked peptides

• Map interaction interfaces at amino acid resolution

• This approach is particularly valuable for membrane protein complexes

4. Proximity-based Labeling:

• Generate CCMFN2 fusion with BioID or APEX2 proximity labeling enzymes

• Express in plant systems and activate labeling

• Isolate biotinylated proteins using streptavidin pulldown

• Identify neighboring proteins by mass spectrometry

• This method captures both stable and transient interactions

5. Bimolecular Fluorescence Complementation (BiFC):

• Create fusion constructs of CCMFN2 and candidate partners with split fluorescent protein fragments

• Express in plant cells and visualize interactions through reconstituted fluorescence

• Include appropriate controls to rule out false positives

• Particularly useful for confirming direct interactions with AtCCMH (AT1G15220)

6. Structural Analysis Approaches:

• Apply cryo-electron microscopy to the purified 500-KDa complex

• Use hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Perform small-angle X-ray scattering for low-resolution structural information

• Combine with computational modeling to generate interaction models

These complementary approaches provide a comprehensive strategy for dissecting CCMFN2's interactions within the 500-KDa complex, potentially revealing its precise role in cytochrome c biogenesis and heme lyase activity.

How can CRISPR-Cas9 gene editing be optimized for studying CCMFN2 function in plant models?

Implementing CRISPR-Cas9 gene editing for CCMFN2 functional studies requires strategic planning and optimization:

1. Guide RNA Design Considerations:

• Target conserved exons early in the CCMFN2 coding sequence to ensure loss-of-function

• Design multiple sgRNAs (3-4) targeting different regions to increase editing efficiency

• Avoid regions with secondary structure that might impede Cas9 binding

• Check for off-target effects using plant genome-specific prediction tools

• Consider the GC content (40-60% ideal) and avoid poly-T sequences (terminate RNA polymerase III transcription)

2. Delivery Methods for Plant Systems:

• Agrobacterium-mediated transformation for most model plants (Arabidopsis)

• Protoplast transfection for rapid validation of editing efficiency

• Biolistic bombardment for recalcitrant species

• RNA-based delivery systems for transient editing experiments

3. Validation Strategies:

• PCR amplification and sequencing of target region

• T7 Endonuclease I assay to detect mutations

• High Resolution Melting Analysis (HRMA) for rapid screening

• Next-generation sequencing for comprehensive mutation profiling

• Western blotting to confirm protein knockout using CCMFN2 antibodies

4. Phenotypic Analysis Pipeline:

• Assess mitochondrial morphology using TEM or fluorescence microscopy

• Measure oxygen consumption rates to evaluate respiratory function

• Analyze cytochrome c content and maturation status

• Evaluate growth under conditions requiring mitochondrial function

• Perform transcriptomics and proteomics to identify compensatory responses

5. Controls and Rescue Experiments:

• Include non-targeting sgRNA controls

• Create heterozygous and homozygous mutant lines for dosage effect studies

• Perform complementation with wildtype CCMFN2 to confirm phenotype specificity

• Consider introducing CCMFN2 variants to identify critical functional domains

6. Species-Specific Considerations:

• For Arabidopsis: Use floral dip transformation for germline editing

• For crop species: Optimize transformation protocols based on established methods

• Consider using multiplex editing to target CCMFN2 homologs in polyploid species

• Evaluate potential effects on agronomic traits if working with crop species

This comprehensive approach enables precise genetic dissection of CCMFN2 function in various plant systems, potentially revealing its exact role in the postulated 500-KDa complex with heme lyase activity and broader implications for mitochondrial bioenergetics.

What imaging methods provide the best resolution for studying CCMFN2 localization within mitochondria?

Optimal imaging of CCMFN2 requires advanced microscopy techniques that overcome the challenges of visualizing mitochondrial membrane proteins:

1. Super-Resolution Microscopy Approaches:

2. Sample Preparation Strategies:

• Optimize fixation protocols to preserve mitochondrial ultrastructure (2-4% paraformaldehyde with 0.05-0.1% glutaraldehyde)

• For plant samples, include cell wall digestion steps (pectolyase, cellulase) to improve antibody penetration

• Use detergent concentrations that permeabilize membranes without disrupting mitochondrial structure

• Consider epitope retrieval methods that maintain spatial organization

3. Labeling Approaches:

• Primary antibody labeling using CCMFN2-specific antibodies

• Secondary antibody selection based on imaging modality (bright organic dyes for STED/STORM)

• Combination with established mitochondrial markers (TOM20, MitoTracker) for context

• For live-cell applications, consider epitope-tagged CCMFN2 constructs

4. Correlative Light and Electron Microscopy (CLEM):

• Perform immunofluorescence imaging of CCMFN2

• Process the same sample for transmission electron microscopy

• Correlate fluorescence signal with ultrastructural features

• Particularly valuable for precise localization within mitochondrial subcompartments

5. Proximity Labeling for Spatial Proteomics:

• Generate APEX2 or TurboID fusions with CCMFN2

• Activate labeling to biotinylate proteins in close proximity

• Visualize biotinylated proteins using fluorescent streptavidin

• Alternatively, isolate and identify proximal proteins by mass spectrometry

6. Advanced Analysis Methods:

• Deconvolution to improve image quality and resolution

• Colocalization analysis with mitochondrial markers

• Quantitative analysis of CCMFN2 distribution patterns

• 3D reconstruction for comprehensive spatial understanding

These imaging approaches can reveal CCMFN2's precise localization within the mitochondrial membrane and its spatial relationship with other components of the 500-KDa complex, providing insights into its role in cytochrome c biogenesis.

How should researchers design experiments to elucidate the proposed heme lyase activity of the CCMFN2-containing complex?

Investigating the proposed heme lyase activity of the CCMFN2-containing complex requires rigorous biochemical and functional approaches:

1. Biochemical Activity Assays:

• Enzyme Activity Measurement: Develop in vitro assays to measure the covalent attachment of heme to cytochrome c apoprotein

• Substrate Preparation: Express and purify cytochrome c apoprotein (without heme)

• Reaction Conditions: Test various buffers, pH conditions, and cofactors that might influence heme lyase activity

• Detection Methods: Use spectrophotometric analysis (absorbance at 550 nm for holo-cytochrome c), fluorescence spectroscopy, or HPLC analysis

2. Complex Isolation and Reconstitution:

• Gentle Extraction: Solubilize mitochondrial membranes using mild detergents (digitonin, n-dodecyl β-D-maltoside)

• Affinity Purification: Use CCMFN2 antibodies for immunoprecipitation of the intact complex

• Size Exclusion Chromatography: Confirm the 500-KDa complex size and assess compositional homogeneity

• In vitro Reconstitution: Attempt to reconstitute the complex from individually purified components

3. Structure-Function Analysis:

• Mutational Studies: Generate point mutations in conserved regions of CCMFN2

• Deletion Analysis: Create truncated versions of CCMFN2 to identify functional domains

• Complementation Experiments: Express mutant versions in CCMFN2-knockout backgrounds

• Activity Correlation: Correlate structural changes with alterations in heme lyase activity

4. Substrate Specificity Determination:

• Variant Substrates: Test activity with different cytochrome c variants

• Heme Analogs: Assess incorporation of modified heme molecules

• Competition Assays: Use structural analogs to identify key recognition features

5. Inhibitor Studies:

• Chemical Inhibition: Screen for compounds that specifically inhibit the heme lyase activity

• Mechanism-Based Inhibitors: Design inhibitors based on transition state analogs

• Antibody Inhibition: Test if specific antibodies against CCMFN2 domains can inhibit activity

6. Multimethod Validation:

• Mass Spectrometry: Detect covalent heme attachment to cytochrome c

• Electron Paramagnetic Resonance: Characterize heme environment before and after attachment

• X-ray Absorption Spectroscopy: Analyze the coordination environment of the iron in heme

• Hydrogen-Deuterium Exchange: Identify conformational changes during catalysis

7. Physiological Relevance:

• Respiratory Chain Assessment: Measure impact of CCMFN2 disruption on electron transport chain function

• Mitochondrial Function Tests: Assess oxygen consumption rates in CCMFN2 mutants

• Cytochrome c Maturation: Quantify mature cytochrome c levels in response to CCMFN2 manipulation

This comprehensive experimental approach will provide multiple lines of evidence to confirm or refute the postulated heme lyase activity of the CCMFN2-containing complex, offering insights into its role in cytochrome c biogenesis and mitochondrial function.

What are the most common technical challenges when working with CCMFN2 antibodies and how can they be addressed?

Researchers working with CCMFN2 antibodies often encounter several technical challenges that can be systematically addressed:

1. Low Signal Intensity:

2. High Background and Non-specific Binding:

3. Antibody Cross-reactivity Issues:

4. Sample Preparation Considerations:

5. Plant-Specific Challenges:

By systematically addressing these challenges, researchers can significantly improve the reliability and reproducibility of experiments using CCMFN2 antibodies across different plant species and experimental conditions .

What controls are essential when investigating CCMFN2 involvement in the cytochrome c biogenesis pathway?

A robust experimental design for studying CCMFN2's role in cytochrome c biogenesis requires comprehensive controls:

1. Antibody Validation Controls:

2. Experimental Controls for Functional Studies:

3. Biochemical Assay Controls:

4. Protein Interaction Controls:

5. Phenotypic Analysis Controls:

6. Mass Spectrometry Controls:

How might active learning approaches be applied to optimize CCMFN2 antibody-antigen binding prediction?

Active learning strategies could significantly enhance CCMFN2 antibody research through computational prediction of binding interactions:

1. Application of Machine Learning to CCMFN2 Antibody Research:

Recent advances in active learning for antibody-antigen binding prediction can be applied to CCMFN2 research. Novel active learning strategies have demonstrated the ability to reduce the number of required antigen mutant variants by up to 35% while accelerating the learning process by 28 steps compared to random sampling baselines . These approaches could be particularly valuable for CCMFN2 antibody development given its conservation across multiple plant species .

2. Implementation Strategy for CCMFN2-Specific Models:

3. Technical Implementation Considerations:

• Feature Engineering: Develop representations capturing both antibody and CCMFN2 sequence/structure features

• Model Architecture: Implement deep learning models capable of handling many-to-many relationships between antibodies and antigens

• Validation Strategy: Use library-on-library screening approaches to validate predictions at scale

• Computational Resources: Leverage distributed computing to handle complex simulation frameworks similar to Absolut!

4. Experimental Validation Protocol:

• In silico prediction of binding between CCMFN2 variants and candidate antibodies

• Prioritization of experiments based on active learning recommendations

• Laboratory validation of highest-priority predictions

• Model updating with new experimental data

• Iterative refinement through additional prediction-validation cycles

5. Potential Benefits for CCMFN2 Research:

• Resource Optimization: Reduce experimental costs by focusing on informative experiments

• Improved Specificity: Design antibodies with enhanced specificity for particular CCMFN2 variants

• Cross-Reactivity Prediction: Accurately predict antibody performance across plant species

• Novel Epitope Discovery: Identify previously unrecognized epitopes for improved antibody development

• Functional Insight: Correlate binding patterns with functional domains in the CCMFN2 protein

This integration of active learning approaches with traditional antibody research could significantly accelerate CCMFN2 studies while reducing experimental costs. The ability to predict antibody performance across the diverse plant species expressing CCMFN2 would be particularly valuable for comparative studies of mitochondrial function in different agricultural and model plant systems.

What are the potential roles of CCMFN2 in plant stress responses and how can they be investigated?

CCMFN2's mitochondrial location suggests potential involvement in plant stress responses, which can be systematically investigated:

1. Theoretical Framework for CCMFN2 in Stress Biology:

Mitochondria play crucial roles in plant stress responses through energy production, redox balance, and retrograde signaling. CCMFN2's presence in the 500-KDa complex with postulated heme lyase activity positions it at the intersection of cytochrome c biogenesis and cellular respiration. This is particularly significant because cytochrome c has dual functions in both electron transport and stress-induced programmed cell death.

2. Comprehensive Research Approach:

3. Stress-Specific Experimental Designs:

4. Multi-omics Integration Strategy:

• Transcriptomics: Compare CCMFN2 expression patterns with known stress-responsive genes

• Proteomics: Identify post-translational modifications on CCMFN2 during stress

• Interactomics: Map stress-specific changes in CCMFN2 protein interaction network

• Metabolomics: Link CCMFN2 function to metabolic adjustments during stress

• Phenomics: Correlate CCMFN2 expression/activity with whole-plant stress phenotypes

5. Comparative Approaches Across Plant Species:

Given CCMFN2's conservation across diverse plant species , comparative studies could reveal:

• Species-specific adaptations in CCMFN2 function related to ecological niches

• Correlation between CCMFN2 sequence variation and stress tolerance capabilities

• Evolutionary conservation of stress-response mechanisms involving CCMFN2

6. Applied Research Directions:

• Crop Improvement: Evaluate CCMFN2 as potential target for enhancing stress tolerance

• Stress Biomarkers: Assess CCMFN2 activity/modification as indicator of mitochondrial stress

• Precision Agriculture: Develop diagnostics based on CCMFN2 status to detect early stress

This research framework would comprehensively address CCMFN2's potential roles in plant stress responses, leveraging its conserved nature across species while focusing on its specific function in mitochondrial complexes involved in energy metabolism and potentially stress signaling.

What are the most critical unresolved questions regarding CCMFN2 function that require future research?

Several fundamental questions about CCMFN2 remain unanswered and represent critical areas for future investigation:

1. Molecular Function Clarification:

• What is the precise enzymatic function of CCMFN2 within the 500-KDa complex?

• Is the postulated heme lyase activity directly catalyzed by CCMFN2 or does it serve as a scaffold protein?

• Which domains or residues are essential for CCMFN2's function in cytochrome c biogenesis?

2. Complex Composition and Dynamics:

• What is the complete protein composition of the 500-KDa complex containing CCMFN2?

• How is assembly of this complex regulated during development and in response to environmental changes?

• What is the stoichiometry of CCMFN2 and AtCCMH within the complex?

3. Regulation and Integration with Cellular Processes:

• How is CCMFN2 expression and activity regulated at the transcriptional, translational, and post-translational levels?

• Does CCMFN2 function change during different developmental stages or in response to mitochondrial stress?

• How is CCMFN2 integrated into broader mitochondrial biogenesis and quality control pathways?

4. Evolutionary Aspects:

• Why is CCMFN2 so highly conserved across diverse plant species , and are there functional differences between orthologs?

• How did the 500-KDa complex evolve, and are there structural or functional homologs in non-plant organisms?

• What selective pressures have maintained CCMFN2 throughout plant evolution?

5. Physiological Significance:

6. Technological Challenges:

• How can we improve methods for studying membrane protein complexes like those containing CCMFN2?

• What new approaches could help visualize the dynamic interactions within these complexes?

• How can we develop more specific tools for manipulating CCMFN2 function in vivo?