CCDC59 Antibody

What are the validated applications for CCDC59 antibody?

The CCDC59 polyclonal antibody (26387-1-AP) has been validated for multiple research applications including Western Blot (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), and ELISA. Each application requires specific optimization for optimal results . For example:

These applications allow researchers to investigate CCDC59 expression, localization, and interactions in experimental systems with human samples .

What is the molecular weight of CCDC59 and how does this impact experimental design?

CCDC59 has an observed molecular weight of 34-38 kDa in experimental conditions . This information is critical when designing Western blot experiments, as researchers should optimize gel percentage and running conditions for proteins in this size range. For optimal resolution of CCDC59, a 10-12% polyacrylamide gel is typically recommended when using standard SDS-PAGE techniques . Additionally, researchers should be aware that post-translational modifications or alternative splicing may cause the protein to migrate at different molecular weights in certain cell types or experimental conditions .

How should antigen retrieval be performed for IHC applications with CCDC59 antibody?

For optimal immunohistochemical staining with CCDC59 antibody (26387-1-AP), heat-mediated antigen retrieval is recommended. Two buffer systems have been validated :

• Primary recommendation: TE buffer at pH 9.0

• Alternative approach: Citrate buffer at pH 6.0

The effectiveness of these protocols has been validated in human lung cancer tissue samples . When performing IHC, researchers should implement the following methodology:

• Use paraffin-embedded tissue sections

• Perform heat-mediated antigen retrieval with the recommended buffers

• Apply the antibody at a dilution of 1:200 (or within the range of 1:50-1:500)

• Include appropriate positive and negative controls for validation

How can I ensure specificity when designing experiments with CCDC59 antibody?

Ensuring antibody specificity is critical for generating reliable research data. For CCDC59 antibody (26387-1-AP), consider implementing these methodological approaches:

• Include proper controls in each experiment:

  • Positive control samples with known CCDC59 expression (e.g., MCF-7 cells for WB, HepG2 cells for IP)

  • Negative controls using either CCDC59-knockout samples or isotype control antibodies

• Validate results across multiple applications:

  • Confirm WB findings with IHC localization studies

  • Support cellular expression with immunoprecipitation data

• Verify antibody specificity:

  • Consider using a second antibody targeting a different epitope of CCDC59

  • Implement siRNA knockdown or CRISPR-based approaches to demonstrate specificity

What strategies can overcome poor signal-to-noise ratio in CCDC59 detection experiments?

When experiencing high background or weak signal in CCDC59 detection, implement these methodological solutions:

For Western Blot applications:

• Optimize blocking conditions (try 5% non-fat milk or 3-5% BSA in TBST)

• Adjust antibody concentration (test dilutions between 1:1000-1:4000)

• Increase washing duration and frequency (4-5 washes of 5-10 minutes each)

• Consider alternative membrane types (PVDF vs. nitrocellulose)

• Experiment with enhanced chemiluminescence detection systems

For Immunohistochemistry:

• Optimize antigen retrieval conditions (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Titrate antibody concentration (between 1:50-1:500)

• Extend blocking time to reduce non-specific binding

• Implement peroxidase blocking steps

• Consider amplification systems for detecting low-abundance targets

For Immunoprecipitation:

• Adjust antibody amount (test range from 0.5-4.0 μg)

• Optimize lysate concentration (1.0-3.0 mg of total protein)

• Consider pre-clearing lysates with protein A/G beads

• Optimize washing buffer stringency

How can I adapt experimental protocols for detecting CCDC59 in different tissue or cell types?

When adapting CCDC59 antibody protocols to new experimental systems, implement this systematic approach:

• Start with validated conditions:

  • For WB: Begin with MCF-7 cell protocols (1:2000 dilution)

  • For IHC: Use human lung cancer tissue parameters as reference (1:200 dilution)

  • For IP: Reference HepG2 cell protocols (4 μg antibody for 4000 μg lysate)

• Optimize for your specific sample:

  • Adjust lysis buffers based on subcellular localization of CCDC59

  • Modify antigen retrieval conditions for different tissue types

  • Test multiple antibody concentrations to determine optimal signal-to-noise ratio

• Implement tissue/cell-specific controls:

  • Include positive control samples where CCDC59 expression is established

  • Run parallel experiments with housekeeping proteins to normalize expression

  • Consider tissue-specific protein interactions that might affect detection

This methodical approach ensures successful protocol transfer while accounting for biological variability across experimental systems.

How should storage and handling conditions be optimized for CCDC59 antibody experiments?

Proper storage and handling significantly impact experimental reproducibility. For CCDC59 antibody, implement these evidence-based practices:

Storage conditions:

• Store at -20°C for long-term stability (stable for one year after shipment)

• Use storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting is not necessary for -20°C storage, though may be preferred for frequently used samples

Working conditions:

• Thaw aliquots completely before use and mix gently to ensure homogeneity

• Avoid repeated freeze-thaw cycles

• For diluted working solutions, prepare fresh or store at 4°C for short periods (1-2 weeks)

• Some preparations may contain 0.1% BSA, which contributes to stability

These practices ensure maintained immunoreactivity and experimental consistency across multiple studies.

What experimental controls should be implemented when studying protein-protein interactions involving CCDC59?

When investigating CCDC59 interactions through techniques like co-immunoprecipitation or proximity ligation assays, implement these control strategies:

• Negative controls:

  • IgG isotype control for non-specific binding

  • Reverse IP with antibodies against putative interacting partners

  • Lysates from CCDC59-depleted cells

• Positive controls:

  • Known protein interactions as methodological controls

  • Reciprocal co-IP experiments (IP with anti-CCDC59 and blot for partner, then reverse)

• Specificity controls:

  • Competition with recombinant CCDC59 protein

  • Validation with multiple antibodies targeting different CCDC59 epitopes

  • Confirmation across multiple experimental systems

• Technical controls:

  • Input samples (5-10% of pre-IP lysate)

  • Beads-only controls to assess non-specific binding

  • Antibody-only controls without cell lysate

This comprehensive control strategy ensures that identified interactions are specific and physiologically relevant.

How can I design experiments to investigate the role of CCDC59 in specific cellular pathways?

To investigate CCDC59's role in cellular pathways, implement this systematic experimental approach:

• Expression analysis workflow:

  • Baseline expression profiling using WB and IHC across relevant cell types/tissues

  • Subcellular localization through fluorescent microscopy using validated antibodies

  • Expression changes under various physiological or stress conditions

• Loss/gain-of-function studies:

  • siRNA/shRNA knockdown with CCDC59 antibody validation of depletion

  • CRISPR-Cas9 knockout with complementation studies

  • Overexpression systems with tagged constructs for functional rescue

• Interaction studies:

  • Co-immunoprecipitation with CCDC59 antibodies (0.5-4.0 μg for 1.0-3.0 mg protein)

  • Proximity ligation assays for in situ interaction detection

  • Yeast two-hybrid or mass spectrometry for unbiased interactome mapping

• Functional readouts:

  • Pathway-specific reporter assays

  • Phosphorylation state analysis of pathway components

  • Cellular phenotype assessment (proliferation, migration, differentiation)

This systematic approach allows robust characterization of CCDC59's functional role in specific pathways through multiple complementary methodologies.

How should I quantify and normalize CCDC59 expression in complex tissue samples?

Accurate quantification of CCDC59 in heterogeneous samples requires careful analytical approaches:

For Western Blot quantification:

• Use appropriate loading controls (GAPDH, β-actin, or tissue-specific stable proteins)

• Implement replicate analysis (minimum n=3) with statistical evaluation

• Consider using fluorescent secondary antibodies for broader linear dynamic range

• Normalize CCDC59 signal to total protein using stain-free technology or Ponceau staining

• Apply densitometry with background subtraction for each lane

For IHC quantification in tissue samples:

• Implement digital image analysis with appropriate thresholding

• Use H-score or Allred scoring systems for semi-quantitative analysis

• Account for tissue heterogeneity with region-of-interest analysis

• Include multiple fields per sample (minimum 5-10 high-power fields)

• Conduct pathologist-blinded scoring to reduce bias

For comparing across experimental conditions:

• Apply appropriate statistical tests (ANOVA with post-hoc analysis for multiple comparisons)

• Report data as fold-change relative to control samples

• Include error metrics (standard deviation or standard error of mean)

These approaches ensure rigorous quantification that accounts for technical and biological variation in complex samples.

What are the key considerations when interpreting CCDC59 antibody data across multiple experimental platforms?

When integrating CCDC59 data from complementary techniques (WB, IHC, IP), consider these analytical principles:

• Platform-specific limitations:

  • WB provides information on protein size and abundance but loses spatial context

  • IHC provides localization but may have lower specificity and limited quantification

  • IP reveals interactions but may detect non-physiological associations

• Concordance analysis:

  • Evaluate consistency across platforms (e.g., whether high WB expression correlates with strong IHC staining)

  • Investigate discrepancies systematically (e.g., if IHC shows nuclear localization but fractionation shows cytoplasmic enrichment)

  • Consider isoform-specific detection differences between techniques

• Contextual interpretation:

  • Compare findings with available literature on CCDC59

  • Consider cell type-specific or context-dependent regulation

  • Evaluate findings in appropriate physiological or pathological contexts

• Technical validation:

  • Confirm antibody lot consistency for longitudinal studies

  • Implement standardized positive controls across experiments

  • Document all technical parameters for reproducibility

How can I assess potential non-specific binding or cross-reactivity in my CCDC59 antibody experiments?

To systematically evaluate and address potential non-specific binding:

• Experimental validation approaches:

  • Pre-absorption controls with recombinant CCDC59 protein

  • Peptide competition assays with immunizing peptide

  • Comparison of staining patterns across multiple CCDC59 antibodies

  • Analysis in CCDC59 knockout/knockdown systems

• Bioinformatic analysis:

  • Sequence homology screening against related proteins

  • Epitope uniqueness assessment through database searches

  • Evaluation of potential post-translational modifications affecting specificity

• Signal verification strategies:

  • Size validation in Western blots (expected 34-38 kDa band)

  • Subcellular localization consistency with known CCDC59 biology

  • Correlation of signal intensity with orthogonal measures of CCDC59 expression

• Specialized controls:

  • Isotype control experiments at equivalent concentrations

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

  • Analysis in tissues/cells with known negative expression

These systematic approaches allow researchers to confidently distinguish specific CCDC59 signal from potential artifacts or cross-reactivity.

How can CCDC59 antibody be used in conjunction with other techniques for studying protein-protein interaction networks?

Integrating CCDC59 antibody-based approaches with complementary techniques creates a powerful framework for interaction network analysis:

• Primary interaction detection:

  • Co-immunoprecipitation with CCDC59 antibody (0.5-4.0 μg)

  • Proximity ligation assay for in situ visualization of interactions

  • FRET/BRET approaches with fluorescently tagged constructs

• Network mapping extensions:

  • Mass spectrometry after CCDC59 immunoprecipitation

  • BioID or APEX proximity labeling with CCDC59 fusion proteins

  • Yeast two-hybrid screening with CCDC59 as bait

• Functional validation:

  • Mutational analysis of interaction domains

  • Competitive peptide inhibition based on interaction interfaces

  • Cellular assays measuring functional consequences of disrupted interactions

• Visualization approaches:

  • Multi-color immunofluorescence for co-localization analysis

  • Super-resolution microscopy for nanoscale interaction assessment

  • Live-cell imaging with tagged constructs to assess dynamics

This multi-technique approach provides complementary layers of evidence for interaction networks, combining the specificity of antibody-based detection with the breadth and functional insights of other methodologies.

What methodological approaches can help distinguish between different isoforms or post-translationally modified forms of CCDC59?

To effectively differentiate CCDC59 variants in experimental systems:

• Electrophoretic separation strategies:

  • High-resolution SDS-PAGE (12-15% gels) for closely migrating isoforms

  • Phos-tag gels for separating phosphorylated variants

  • 2D gel electrophoresis for complex post-translational modification patterns

• Antibody-based discrimination:

  • Use of isoform-specific antibodies when available

  • Phospho-specific antibodies for key modification sites

  • Sequential immunoprecipitation to isolate specific CCDC59 subpopulations

• Mass spectrometry approaches:

  • Targeted MS after immunoprecipitation with CCDC59 antibody

  • Parallel reaction monitoring for specific modified peptides

  • SILAC labeling for quantitative analysis of modification dynamics

• Functional characterization:

  • Isoform-specific knockdown/rescue experiments

  • Site-directed mutagenesis of modification sites

  • Subcellular fractionation to assess localization differences between variants

These methodological approaches allow researchers to move beyond total CCDC59 detection to understand the functional diversity arising from different protein variants.

How might advances in antibody design, such as paratope-mimicking peptides, impact future CCDC59 research?

The development of antibody-mimicking technologies, as demonstrated with CD59 antibodies, represents a frontier with significant implications for CCDC59 research :

Emerging methodological approaches include:

• Computational design of CCDC59-targeting molecules:

  • Bioinformatic prediction of epitope regions

  • Molecular modeling of antibody-antigen interactions

  • Rational design of paratope-mimicking structures

• Alternative binding scaffolds:

  • Development of bicyclic peptides targeting key CCDC59 epitopes

  • Single-domain antibody fragments with enhanced tissue penetration

  • Aptamer-based detection systems for live-cell applications

• Functional extensions:

  • Bispecific molecules targeting CCDC59 and interacting partners

  • Cell-permeable variants for intracellular targeting

  • Peptide-drug conjugates for targeted manipulation of CCDC59 function

These advances promise to expand the toolkit beyond traditional antibodies, potentially offering enhanced specificity, reduced production costs, and novel functional applications in CCDC59 research.

What are the current limitations in CCDC59 antibody research and how might they be addressed in future studies?

Current methodological limitations and future directions include:

• Current technical challenges:

  • Limited availability of CCDC59 antibodies targeting different epitopes

  • Incomplete validation across diverse experimental systems and species

  • Potential cross-reactivity with structurally related coiled-coil proteins

  • Limited information on epitope mapping and binding characteristics

• Future methodological solutions:

  • Development of monoclonal antibodies with defined epitopes

  • Comprehensive cross-validation across multiple antibody sources

  • Enhanced validation in knockout systems using CRISPR-Cas9 technology

  • Implementation of standardized reporting guidelines for antibody validation

• Emerging technological approaches:

  • Single-cell analysis methods to assess heterogeneity in CCDC59 expression

  • In situ detection systems with enhanced sensitivity and specificity

  • Integrated multi-omics approaches linking CCDC59 protein data with transcriptomic profiles

  • Advanced imaging modalities for dynamic analysis of CCDC59 in living systems