C2CD5 Antibody

What is C2CD5 and what biological functions has it been associated with?

C2CD5, also known as C2 domain-containing phosphoprotein (CDP138), is a protein involved in cellular trafficking processes with significant implications for metabolic regulation. Recent studies have established that C2CD5 plays a critical role in mitochondrial function, particularly within hypothalamic neurons .

The protein has been linked to energy homeostasis regulation, as mice lacking C2CD5 develop obesity and display reduced functional melanocortin receptor 4 (MC4R) at the surface of hypothalamic neurons . This suggests C2CD5 is involved in receptor trafficking pathways that regulate energy balance. Loss of C2CD5 alters mitochondrial ultrastructure, localization, and activity within hypothalamic neurons, positioning this protein as a potential link between hypothalamic dysfunction, mitochondrial abnormalities, and obesity .

What types of C2CD5 antibodies are currently available for research applications?

Based on available information, researchers can access rabbit polyclonal antibodies against human C2CD5 . These antibodies are typically provided at concentrations of approximately 0.05 mg/ml and are manufactured using standardized processes to ensure rigorous quality standards .

When selecting a C2CD5 antibody for your research, consider these critical factors:

• Species reactivity (human, mouse, etc.)

• Clonality (polyclonal vs. monoclonal)

• Applications validated for (IHC, WB, ICC-IF)

• Epitope recognition region

• Validation methodology used by manufacturer

What validation methods are employed to ensure C2CD5 antibody specificity?

C2CD5 antibodies undergo multiple validation approaches to ensure specificity and reliability across different experimental contexts. Standard validation methods include :

• Immunohistochemistry (IHC) - Testing on tissue sections with known expression patterns

• Immunocytochemistry/Immunofluorescence (ICC-IF) - Evaluating cellular localization patterns

• Western Blot (WB) - Confirming specificity by molecular weight detection

Enhanced validation methods may also be employed:

• Genetic validation using knockout or knockdown models

• Independent antibody validation using antibodies against different epitopes

• Orthogonal validation correlating with mRNA expression data

• Physiological validation under different biological conditions or treatments

These comprehensive validation approaches help ensure the antibody specifically recognizes C2CD5 and performs consistently across experimental settings.

How should researchers design experiments using C2CD5 antibodies in studies of hypothalamic function?

When designing experiments to investigate C2CD5's role in hypothalamic function, researchers should consider these methodological approaches:

• Tissue preparation:

  • Use fresh or properly fixed hypothalamic tissue

  • Consider microdissection of specific nuclei (arcuate, paraventricular)

  • Include positive and negative control tissues

• Experimental controls:

  • Use tissues from C2CD5 knockout models as negative controls

  • Include isotype controls to assess non-specific binding

  • Consider peptide competition assays to confirm specificity

• Multi-method approach:

  • Combine protein detection (immunoblotting, immunostaining) with functional assays

  • Correlate findings with behavioral or metabolic phenotypes

  • Consider co-localization studies with mitochondrial markers

• Sex-specific considerations:

  • Include both male and female experimental subjects

  • Analyze data for potential sex-specific differences

  • Control for hormonal variations that may affect results

What are the recommended storage and handling protocols for C2CD5 antibodies?

While specific protocols may vary between manufacturers, standard guidelines for rabbit polyclonal antibodies against C2CD5 typically include:

• Storage conditions:

  • Store at -20°C for long-term preservation

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Short-term storage (1-2 weeks) at 4°C is generally acceptable

• Buffer composition:

  • Typically supplied in buffered aqueous glycerol solutions

  • Buffer composition designed to maintain antibody stability and activity

  • Preservatives may be included to prevent microbial contamination

• Working solutions:

  • Prepare fresh working dilutions on the day of experiment

  • Determine optimal dilutions empirically for each application

  • Return stock solutions to recommended storage temperature promptly

• Quality control:

  • Monitor performance through consistent positive controls

  • Document lot numbers and maintain validation records

  • Consider stability testing for antibodies stored long-term

How can researchers optimize C2CD5 antibody protocols for detecting mitochondrial abnormalities?

Given C2CD5's role in mitochondrial function within hypothalamic neurons , optimizing antibody protocols for this specific application requires sophisticated approaches:

Methodology for Mitochondrial Co-localization Studies:

• Sample preparation optimization:

  • Use gentle fixation methods to preserve mitochondrial ultrastructure

  • Consider live-cell imaging for dynamic trafficking studies

  • Optimize permeabilization to balance antibody access with structural preservation

• Advanced imaging strategies:

  • Employ super-resolution microscopy (STED, STORM) to resolve mitochondrial details

  • Use multi-color confocal microscopy with spectral unmixing

  • Conduct time-lapse imaging to capture trafficking dynamics

• Quantitative analysis methods:

  • Measure mitochondrial morphology parameters (size, aspect ratio, branching)

  • Quantify mitochondrial distribution relative to cellular compartments

  • Analyze co-localization coefficients with mitochondrial markers

• Validation approaches:

  • Compare findings with electron microscopy data

  • Correlate antibody staining with functional mitochondrial assays

  • Confirm specificity using siRNA knockdown or CRISPR knockout models

What methodological considerations apply when using C2CD5 antibodies in obesity research?

The established connection between C2CD5 deficiency and obesity in mouse models presents unique methodological considerations:

• Experimental design strategies:

  • Compare lean versus obese models for C2CD5 expression/localization differences

  • Analyze changes in C2CD5 distribution following metabolic challenges

  • Conduct time-course studies to capture dynamic changes

• Tissue-specific optimization:

  • Modify protocols for high-lipid content tissues (hypothalamus, adipose)

  • Adjust blocking parameters to reduce background in metabolically active tissues

  • Consider tissue clearing techniques for thick-section 3D imaging

• Functional correlation methods:

  • Correlate C2CD5 immunostaining with MC4R trafficking data

  • Examine relationship between C2CD5 localization and mitochondrial function

  • Quantify associations between C2CD5 levels and metabolic parameters

• Methodological controls:

  • Include age-matched and diet-matched controls

  • Account for circadian variations in metabolic tissues

  • Use tissues from both sexes to identify potential dimorphic responses

How do C2CD5 knockout models compare with antibody neutralization for functional studies?

Understanding the advantages and limitations of different approaches is critical for robust experimental design:

Comparative Analysis of C2CD5 Study Methods:

• C2CD5 Knockout Models:

  • Advantages:

    • Complete absence of protein for clear phenotypic analysis

    • Ability to study developmental effects and compensatory mechanisms

    • Established obesity phenotype with altered MC4R trafficking

  • Limitations:

    • Potential developmental compensation by related proteins

    • Inability to study temporal aspects of protein function

    • Challenging to distinguish direct from indirect effects

• Antibody-Based Approaches:

  • Advantages:

    • Can be applied to study acute effects

    • Potential to target specific protein domains or epitopes

    • Applicable across diverse experimental systems

  • Limitations:

    • Incomplete inhibition of protein function

    • Access challenges for intracellular targets

    • Potential cross-reactivity with related proteins

• Combined Approaches:

  • Use knockout models to establish baseline phenotypes

  • Apply antibodies for mechanistic studies in wild-type systems

  • Validate findings across multiple methodological approaches

  • Employ rescue experiments to confirm specificity

What are the potential cross-reactivity concerns with C2CD5 antibodies?

When working with antibodies targeting C2CD5, researchers should consider several potential cross-reactivity issues:

• Family protein cross-reactivity:

  • C2CD5 belongs to the C2 domain-containing protein family

  • Potential cross-reactivity with related proteins like C2CD4C

  • Shared structural domains may lead to non-specific binding

• Tissue-specific considerations:

  • Expression patterns of related proteins vary across tissues

  • Background signal may differ between neural and metabolic tissues

  • Optimization may be required for each tissue type

• Validation approaches:

  • Western blot analysis to confirm single band at expected molecular weight

  • Testing on C2CD5 knockout tissues as negative controls

  • Peptide competition assays to verify epitope specificity

  • Orthogonal validation comparing with mRNA expression data

• Application-specific optimization:

  • Adjust antibody concentration based on application and tissue

  • Modify blocking protocols to reduce non-specific binding

  • Consider using monoclonal antibodies for highly specific applications

How can researchers validate C2CD5 antibody performance in receptor trafficking studies?

Given the relationship between C2CD5 and MC4R trafficking , specialized validation approaches are recommended:

• Co-localization validation:

  • Dual immunolabeling with MC4R and C2CD5 antibodies

  • Super-resolution microscopy to resolve trafficking compartments

  • Quantitative co-localization analysis with appropriate statistical measures

• Functional validation:

  • Surface biotinylation assays to quantify receptor externalization

  • Pulse-chase experiments to track receptor trafficking kinetics

  • FRAP (Fluorescence Recovery After Photobleaching) to measure mobility

• Molecular interaction validation:

  • Co-immunoprecipitation experiments to detect protein complexes

  • Proximity ligation assays (PLA) to confirm close association (<40nm)

  • FRET/BRET analysis for direct interaction studies

• Control experiments:

  • Compare wild-type and C2CD5 knockout conditions

  • Include pharmacological controls (trafficking inhibitors)

  • Perform parallel analysis with known C2CD5 interaction partners

How can researchers address non-specific binding when using C2CD5 antibodies?

Non-specific binding can compromise experimental results when working with C2CD5 antibodies. Consider these methodological solutions:

• Optimization strategies:

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

  • Extend blocking time using 5-10% normal serum from the secondary antibody species

  • Test different detergents (Triton X-100, Tween-20, saponin) for permeabilization

  • Consider using protein-free blockers for high background tissues

• Technical modifications:

  • Increase washing duration and volume between antibody incubations

  • Pre-adsorb antibody with acetone powder from negative control tissues

  • Use monovalent Fab fragments to reduce non-specific binding

  • Consider tyramide signal amplification for low abundance targets

• Control experiments:

  • Include isotype controls at equivalent concentrations

  • Perform secondary-only controls to assess non-specific binding

  • Use C2CD5 knockout tissue sections as negative controls

  • Compare multiple antibodies targeting different C2CD5 epitopes

What approaches can resolve discrepancies in C2CD5 localization patterns?

Researchers may encounter contradictory results regarding C2CD5 subcellular localization. These approaches can help resolve such discrepancies:

• Technical considerations:

  • Compare fixation methods (PFA, methanol, acetone) for effects on epitope accessibility

  • Assess permeabilization impact on subcellular structure preservation

  • Evaluate antibody penetration in different subcellular compartments

  • Consider native versus tagged protein localization studies

• Methodological validation:

  • Fractionate cells and perform Western blot analysis on subcellular fractions

  • Use multiple antibodies targeting different epitopes of C2CD5

  • Correlate antibody findings with fluorescently-tagged C2CD5 localization

  • Employ live-cell imaging to capture dynamic localization patterns

• Biological variables:

  • Assess localization under different metabolic conditions

  • Compare localization across cell types and tissues

  • Examine developmental or activation-dependent changes

  • Consider post-translational modifications affecting localization

How can C2CD5 antibodies be utilized in studies of neuronal mitochondrial dysfunction?

Given C2CD5's role in mitochondrial dynamics , these advanced methodological approaches can enhance research in neuronal systems:

• Mitochondrial morphology analysis:

  • Combine C2CD5 immunostaining with mitochondrial markers (TOMM20, MitoTracker)

  • Quantify mitochondrial fragmentation, elongation, and network complexity

  • Correlate C2CD5 levels with mitochondrial distribution patterns

  • Analyze ultrastructural features using immuno-electron microscopy

• Functional correlation studies:

  • Integrate C2CD5 staining with measurements of mitochondrial membrane potential

  • Correlate C2CD5 distribution with oxygen consumption rates

  • Assess relationship between C2CD5 levels and mitochondrial calcium handling

  • Examine ATP production in relation to C2CD5 expression patterns

• Dynamic trafficking analysis:

  • Perform dual-color live imaging of C2CD5 and mitochondria

  • Measure transport parameters (velocity, distance, directionality)

  • Quantify mitochondrial fusion/fission events relative to C2CD5 localization

  • Analyze recovery kinetics after photobleaching or photoactivation

What methodological approaches are recommended for studying C2CD5 in metabolic disease models?

Researchers investigating C2CD5's role in metabolic disorders should consider these specialized approaches:

• Model-specific considerations:

  • Diet-induced obesity models: Track C2CD5 expression changes during progression

  • Genetic obesity models: Compare C2CD5 localization with wild-type counterparts

  • Conditional knockout approaches: Target C2CD5 deletion in specific metabolic tissues

  • Humanized models: Validate findings across species

• Multi-tissue analysis:

  • Hypothalamic nuclei: Focus on arcuate and paraventricular regions

  • Peripheral tissues: Examine adipose, liver, and muscle C2CD5 expression

  • Blood-brain barrier: Investigate potential transport or signaling roles

  • Circuit-level analysis: Map C2CD5 expression in feeding/satiety circuits

• Functional correlation methods:

  • Metabolic phenotyping: Correlate C2CD5 levels with energy expenditure, food intake

  • Mitochondrial function: Compare respiratory capacity with C2CD5 expression

  • Receptor trafficking: Quantify surface vs. internal MC4R relative to C2CD5 levels

  • Gene expression analysis: Identify transcriptional networks associated with C2CD5

How might C2CD5 antibodies contribute to therapeutic development for metabolic disorders?

Given the established link between C2CD5 deficiency and obesity , antibody-based research may facilitate therapeutic strategies:

• Target validation approaches:

  • Map critical functional domains using epitope-specific antibodies

  • Identify protein interactions amenable to therapeutic disruption

  • Validate relationships between C2CD5 and established metabolic regulators

  • Define tissue-specific functions to guide intervention strategies

• Biomarker development:

  • Assess C2CD5 expression patterns in clinical samples from metabolic disease patients

  • Correlate protein levels or localization with disease severity or progression

  • Investigate post-translational modifications as potential disease markers

  • Develop sensitive detection methods for diagnostic applications

• Therapeutic screening strategies:

  • Use C2CD5 antibodies in high-content screening for compound effects

  • Develop cell-based assays to monitor C2CD5-dependent receptor trafficking

  • Establish in vitro systems to assess mitochondrial function in relation to C2CD5

  • Create reporter assays to track C2CD5 activity in response to interventions