ccdc102a Antibody

What is CCDC102A and why is it studied in research?

CCDC102A (Coiled-Coil Domain Containing 102A) is a protein with gene ontology annotations that include motor activity and association with the myosin complex . The protein's coiled-coil structure suggests potential roles in protein-protein interactions and cellular structural organization. Researchers study CCDC102A to better understand its biological functions in normal physiology and potential implications in disease states. Current research focuses on characterizing its expression patterns across tissues and identifying its interaction partners within cellular complexes.

What types of CCDC102A antibodies are available for research applications?

Several types of CCDC102A antibodies are available for research, with rabbit polyclonal antibodies being the most common . These antibodies target different epitope regions of the CCDC102A protein, including C-terminal regions (amino acids 401-430) . While polyclonal antibodies offer broad epitope recognition, some applications may benefit from the specificity of monoclonal antibodies. Most commercially available antibodies are unconjugated, though some may be available with fluorescent or enzymatic conjugations for specialized applications.

What species reactivity should researchers consider when selecting a CCDC102A antibody?

When selecting a CCDC102A antibody, researchers should carefully consider species reactivity based on their experimental model. Available antibodies demonstrate different reactivity profiles:

• Human-reactive only

• Human and mouse cross-reactive

• Mouse-reactive with predicted bovine cross-reactivity

Species reactivity is determined by sequence homology in the target epitope region. Researchers should examine sequence alignment data between species for the epitope region of interest before selecting an antibody, particularly when working with non-human models.

What are the validated applications for CCDC102A antibodies?

CCDC102A antibodies have been validated for multiple experimental applications, which vary by specific antibody product:

Researchers should optimize dilutions for their specific experimental conditions, as these recommended ranges serve only as starting points.

How should researchers optimize antibody dilution for Western blot applications?

For Western blot optimization with CCDC102A antibodies, researchers should perform a dilution series experiment. Begin with the manufacturer's recommended range (typically 1:500-1:3000) and test multiple dilutions simultaneously against your sample and appropriate controls. Start with a protein loading amount of 20-30 μg of total protein per lane. Evaluate signal-to-noise ratio, background levels, and detection sensitivity at each dilution. For optimal results, include both positive controls (tissues/cells known to express CCDC102A) and negative controls (tissues/cells with minimal expression or knockout samples). Once the optimal dilution is identified, validate reproducibility across multiple experiments.

What are the recommended fixation and antigen retrieval methods for immunohistochemistry with CCDC102A antibodies?

For immunohistochemistry applications, formalin-fixed paraffin-embedded (FFPE) tissues are commonly used with CCDC102A antibodies . Standard fixation involves 10% neutral buffered formalin for 24-48 hours. For antigen retrieval, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is generally recommended, though some epitopes may require EDTA buffer (pH 9.0). Researchers should optimize antigen retrieval time (typically 10-20 minutes) and temperature based on their specific tissue type and fixation conditions. Fresh frozen tissues may provide better epitope preservation but require optimization of fixation protocols (typically 4% paraformaldehyde for 10-15 minutes) prior to antibody incubation.

How should researchers validate CCDC102A antibody specificity?

Validating antibody specificity is critical for reliable CCDC102A research. A comprehensive validation approach includes:

• Knockout/knockdown controls: Compare staining patterns between wild-type and CCDC102A-knockout/knockdown samples. Significant reduction in signal indicates specificity.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before applying to samples. Signal reduction indicates specific binding.

• Multiple antibody comparison: Test multiple antibodies targeting different epitopes of CCDC102A. Consistent localization patterns support specificity.

• Western blot analysis: Confirm detection of a single band at the expected molecular weight (~62.6 kDa) or predicted splice variant weights.

• Recombinant protein controls: Use purified recombinant CCDC102A protein as a positive control in immunoblotting applications.

Proper validation prevents misinterpretation of experimental results due to non-specific binding or cross-reactivity.

What controls should be included in CCDC102A antibody experiments?

Well-designed experiments with CCDC102A antibodies should include multiple controls:

• Positive tissue/cell controls: Samples known to express CCDC102A at detectable levels based on literature reports.

• Negative tissue/cell controls: Samples with minimal CCDC102A expression or knockout models.

• Technical controls:

  • Primary antibody omission control to assess secondary antibody specificity

  • Isotype control (rabbit IgG at equivalent concentration) to assess non-specific binding

  • Loading controls for Western blot (β-actin, GAPDH, α-tubulin)

  • Autofluorescence controls for immunofluorescence applications

• Method validation controls: Include recombinant PrEST antigen CCDC102A as a standard for quantitative applications.

These controls help distinguish specific signals from artifacts and allow for accurate interpretation of experimental results.

How should researchers determine the optimal sample preparation for CCDC102A detection?

Sample preparation significantly impacts CCDC102A detection. For cellular/tissue lysates in Western blot applications, researchers should compare:

• Lysis buffer composition: Test RIPA buffer versus gentler NP-40 or Triton X-100 based buffers to preserve protein structure.

• Denaturing conditions: Compare reducing versus non-reducing conditions, as the coiled-coil domains may be sensitive to reduction.

• Cellular fractionation: Consider separate analysis of cytoplasmic, nuclear, and membrane fractions to determine CCDC102A localization.

• Fixation methods for microscopy: Compare paraformaldehyde, methanol, and acetone fixation for optimal epitope preservation.

• Sample processing timing: Minimize delays between sample collection and processing to prevent protein degradation.

Systematic optimization ensures reliable and reproducible detection of CCDC102A across experimental conditions.

How can researchers address weak or absent CCDC102A signal in Western blot applications?

When encountering weak or absent CCDC102A signal in Western blots, systematically troubleshoot using this approach:

• Protein expression verification: Confirm CCDC102A expression in your sample type through RT-PCR or publicly available expression databases.

• Protein extraction optimization: CCDC102A has predicted motor activity and myosin complex association , suggesting it may be compartmentalized. Test different extraction methods including cytoskeletal extraction buffers.

• Transfer efficiency: Use reversible staining (Ponceau S) to verify successful protein transfer to membrane. For high molecular weight proteins, extend transfer time or reduce SDS concentration.

• Antibody sensitivity: Increase protein loading (up to 50-75 μg), reduce antibody dilution, or extend incubation time/temperature.

• Detection system sensitivity: Switch to more sensitive detection methods like chemiluminescence enhancers or fluorescent secondary antibodies.

• Membrane selection: Compare PVDF (higher protein binding capacity) versus nitrocellulose membranes (lower background).

Document all optimization steps to establish a reliable protocol for future experiments.

How should researchers interpret unexpected bands in CCDC102A Western blots?

When detecting unexpected bands in CCDC102A Western blots, consider these analytical approaches:

• Predicted molecular weight: The calculated molecular weight of CCDC102A is approximately 62.6 kDa . Bands at different molecular weights may represent:

  • Post-translational modifications (phosphorylation, glycosylation)

  • Alternative splice variants

  • Proteolytic fragments

  • Protein complexes (if sample not fully denatured)

  • Non-specific binding

• Validation strategies:

  • Compare band patterns across different antibodies targeting different epitopes

  • Perform immunoprecipitation followed by mass spectrometry to identify the unexpected proteins

  • Test samples with known CCDC102A knockdown/knockout

  • Investigate tissue-specific expression patterns of potential splice variants

• Literature comparison: Search for published Western blot images using CCDC102A antibodies to compare band patterns.

Thorough documentation of unexpected bands may lead to novel discoveries about CCDC102A processing or interactions.

How can researchers minimize background in immunohistochemistry/immunofluorescence with CCDC102A antibodies?

High background is a common challenge in CCDC102A immunostaining. Implement these optimization strategies:

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Triton X-100 for better penetration

• Antibody optimization:

  • Further dilute primary antibody (test range from 1:50-1:500)

  • Reduce incubation temperature (4°C overnight instead of room temperature)

  • Add 0.05% Tween-20 to antibody diluent to reduce non-specific binding

• Washing optimization:

  • Increase wash steps (5-6 washes of 5 minutes each)

  • Add 0.05-0.1% Tween-20 to wash buffers

  • Use TBS instead of PBS if phospho-specific epitopes are targeted

• Tissue-specific considerations:

  • Perform antigen-retrieval optimization for each tissue type

  • Include tissue-specific autofluorescence quenching steps

  • Consider tissue permeabilization optimization

Systematic evaluation of these parameters will help establish optimal conditions for specific signal detection.

How can CCDC102A antibodies be utilized in co-immunoprecipitation studies to identify interaction partners?

For investigating CCDC102A protein interactions using co-immunoprecipitation (Co-IP), researchers should:

• Antibody selection: Choose antibodies demonstrated to recognize native (non-denatured) CCDC102A. Test polyclonal antibodies from different hosts to identify optimal antibody for immunoprecipitation.

• Lysis conditions: Use gentle lysis buffers (150-300 mM NaCl, 1% NP-40 or 0.5% Triton X-100) to preserve protein-protein interactions. Consider crosslinking for transient interactions.

• Experimental design:

  • Include IgG control immunoprecipitation to identify non-specific binding

  • Perform reciprocal Co-IPs with antibodies against predicted interaction partners

  • Compare results from multiple cell types or tissues to identify context-dependent interactions

• Validation approach:

  • Confirm interactions through orthogonal methods (proximity ligation assay, FRET)

  • Perform mass spectrometry analysis of immunoprecipitated complexes

  • Validate key interactions through recombinant protein pull-down assays

These approaches will help identify biologically relevant CCDC102A interaction partners and inform functional studies.

How can researchers use CCDC102A antibodies to investigate subcellular localization and trafficking?

To investigate CCDC102A subcellular localization and potential trafficking, researchers can implement these advanced approaches:

• Multi-channel confocal microscopy: Co-stain with CCDC102A antibody and markers for:

  • Cytoskeletal components (due to predicted motor activity)

  • Organelle markers (Golgi, ER, mitochondria, endosomes)

  • Cell cycle phase-specific markers

• Live-cell imaging strategies:

  • Express fluorescently-tagged CCDC102A and validate localization with antibody staining

  • Perform antibody feeding assays (for membrane-associated pools)

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to assess mobility

• Ultrastructural localization:

  • Use immunogold electron microscopy for precise subcellular localization

  • Perform proximity labeling (BioID, APEX) followed by CCDC102A antibody validation

• Stimulus-dependent trafficking:

  • Investigate localization changes during cell cycle progression

  • Test effects of cytoskeletal disrupting agents (nocodazole, cytochalasin D)

  • Examine responses to cellular stress (oxidative, heat shock, nutrient deprivation)

These approaches provide comprehensive insights into the dynamic behavior and functional contexts of CCDC102A within cells.

What strategies can researchers employ to study post-translational modifications of CCDC102A using available antibodies?

Investigating post-translational modifications (PTMs) of CCDC102A requires specialized approaches:

• PTM prediction and targeting:

  • Use bioinformatic tools to predict potential phosphorylation, acetylation, or ubiquitination sites

  • Generate or obtain modification-specific antibodies for predicted sites

  • Compare migration patterns using standard CCDC102A antibodies before and after treatments that affect PTMs

• Enrichment strategies:

  • Immunoprecipitate CCDC102A using available antibodies followed by PTM-specific antibody detection

  • Use phosphatase/deacetylase treatments to confirm modification-dependent band shifts

  • Employ PTM enrichment techniques (phosphopeptide enrichment, ubiquitin remnant motif antibodies) before mass spectrometry

• Functional validation:

  • Create site-specific mutants (alanine substitutions) and compare localization/function to wild-type

  • Use pharmacological inhibitors of specific modifying enzymes to assess functional consequences

  • Implement temporal analysis during cellular processes (cell cycle, differentiation) to identify regulated modifications

These approaches will provide insights into the regulatory mechanisms controlling CCDC102A function through post-translational modifications.