CCDC153 Antibody

What is CCDC153 and what applications are CCDC153 antibodies validated for?

CCDC153 (coiled-coil domain containing 153) is a protein whose properties remain largely unelucidated. Current research suggests it may function as a neuronal subtype marker according to published literature (PMID: 28166221) . The protein has a calculated molecular weight of 24 kDa (210 amino acids), though it is frequently observed at 45-48 kDa on Western blots, potentially due to dimerization .

CCDC153 antibodies have been validated for multiple applications:

For optimal results, titration in each specific testing system is strongly recommended as optimal concentrations may be sample-dependent .

What species reactivity has been confirmed for commercial CCDC153 antibodies?

Commercial CCDC153 antibodies have demonstrated consistent reactivity across multiple mammalian species. According to validation data from multiple manufacturers, the polyclonal rabbit antibodies against CCDC153 show reactivity with:

• Human samples

• Mouse samples

• Rat samples

It's important to note that while these three species have been confirmed, other species have not been extensively tested or validated . When planning experiments with tissue or cells from species other than human, mouse, or rat, preliminary validation experiments should be conducted to confirm cross-reactivity before proceeding with full-scale studies.

How should CCDC153 antibody be stored to maintain optimal activity?

Proper storage of CCDC153 antibody is essential to maintain its reactivity and specificity. Based on manufacturer recommendations, the following storage conditions should be observed:

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

• The antibody is stable for one year after shipment when stored at -20°C

• Some formulations do not require aliquoting for -20°C storage, but this may vary by manufacturer

• Short-term storage at 4°C is acceptable for some formulations

• The antibody is typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some smaller volume formulations (20μl) may contain 0.1% BSA as a stabilizer

For most research applications, avoid repeated freeze-thaw cycles as these can significantly degrade antibody performance . If frequent use is anticipated, consider preparing working aliquots upon receipt, unless specifically advised against aliquoting by the manufacturer .

How do you explain the discrepancy between calculated (24 kDa) and observed (45-48 kDa) molecular weights of CCDC153?

The discrepancy between the calculated molecular weight of CCDC153 (24 kDa, 210 amino acids) and its observed migration pattern on Western blots (45-48 kDa) represents an interesting biochemical phenomenon that requires careful interpretation . This discrepancy may be explained by several factors:

• Dimerization: The most commonly cited explanation is that CCDC153 forms dimers that remain stable even under the denaturing conditions of SDS-PAGE. Multiple manufacturers specifically note that "the 45-48 kDa band may be a dimer of CCDC153" . This is consistent with the behavior of many coiled-coil domain-containing proteins, which often form stable oligomeric structures.

• Post-translational modifications: Although not explicitly mentioned in the search results, many proteins undergo modifications such as glycosylation, phosphorylation, or ubiquitination that can significantly increase their apparent molecular weight.

• Protein structure effects: Coiled-coil domains can affect protein migration in SDS-PAGE due to their structural properties, sometimes causing anomalous migration patterns.

To experimentally verify the identity of the 45-48 kDa band, researchers should consider:

• Running reducing vs. non-reducing conditions in parallel

• Performing mass spectrometry analysis of the immunoprecipitated protein

• Using alternative antibodies targeting different epitopes of CCDC153

• Conducting knockdown/knockout validation experiments to confirm specificity

Understanding this discrepancy is crucial for accurate interpretation of Western blot results and avoiding misidentification of CCDC153 in experimental systems.

What antigen retrieval methods are optimal for CCDC153 immunohistochemistry in formalin-fixed, paraffin-embedded tissues?

Effective antigen retrieval is critical for successful immunohistochemical detection of CCDC153 in formalin-fixed, paraffin-embedded (FFPE) tissues. Based on validated protocols, the following antigen retrieval methods are recommended:

• Primary recommendation: Tris-EDTA (TE) buffer at pH 9.0

  • This alkaline buffer system has demonstrated superior results in retrieving CCDC153 epitopes in FFPE tissues

  • The higher pH likely facilitates more effective breaking of formalin-induced protein crosslinks

• Alternative option: Citrate buffer at pH 6.0

  • While less optimal than TE buffer, citrate buffer can also effectively retrieve CCDC153 epitopes

  • This method may be preferred when using multiplex IHC with other antibodies that perform better with citrate-based retrieval

For human prostate hyperplasia tissue samples, which have been specifically validated, a dilution range of 1:20-1:200 is recommended following appropriate antigen retrieval . The optimal dilution should be determined empirically for each tissue type.

Validation data from immunohistochemical analysis of paraffin-embedded human prostate hyperplasia tissue demonstrates successful detection using CCDC153 antibody at a dilution of 1:50, with clear visualization under both 10x and 40x magnification . These parameters provide a starting point for optimization in other FFPE tissue types.

What strategies can be employed to validate the specificity of CCDC153 antibody signal in various applications?

Rigorous validation of antibody specificity is essential for generating reliable and reproducible research data. For CCDC153 antibody, which targets a relatively uncharacterized protein, multiple complementary validation approaches are particularly important:

• Positive and negative control cell/tissue selection:

  • Confirmed positive controls: COLO 320 cells, HepG2 cells, L02 cells, human brain tissue, and HeLa cells have been validated for Western blot

  • For immunofluorescence: HepG2 and HeLa cells serve as reliable positive controls

  • Negative controls should include tissues/cells known not to express CCDC153 or isotype control antibodies

• Knockout/knockdown validation:

  • siRNA or CRISPR-based knockout of CCDC153 followed by Western blot analysis

  • This approach provides definitive evidence of antibody specificity by demonstrating loss of signal in genetically modified samples

• Recombinant protein competition assay:

  • Pre-incubate antibody with excess purified CCDC153 recombinant protein

  • Specific binding will be blocked, leading to signal reduction or elimination

• Multiple antibody concordance:

  • Compare results using different antibodies targeting distinct epitopes of CCDC153

  • Consistent results across antibodies increase confidence in specificity

• Immunoprecipitation and mass spectrometry:

  • Perform IP with CCDC153 antibody followed by mass spectrometry analysis

  • This confirms the identity of the immunoprecipitated protein as CCDC153

Some CCDC153 antibodies have undergone specificity verification on protein arrays containing the target protein plus 383 other non-specific proteins , providing additional confidence in their specificity. When establishing a new application or cell/tissue system, researchers should implement at least two independent validation approaches.

What are the optimal protocols for Western blot detection of CCDC153?

Western blot detection of CCDC153 requires careful optimization due to the protein's unique properties, particularly its tendency to appear as a dimer (45-48 kDa) rather than at its calculated molecular weight of 24 kDa . The following protocol incorporates validated parameters for successful CCDC153 detection:

Sample preparation and electrophoresis:

• Extract proteins using standard lysis buffers containing protease inhibitors

• Load 20-40 μg of total protein per lane

• Use 10-12% polyacrylamide gels for optimal resolution of the 45-48 kDa range

• Include molecular weight markers that span 20-75 kDa range to identify both monomeric and dimeric forms

Transfer and antibody incubation:

• Transfer proteins to PVDF or nitrocellulose membrane using standard protocols

• Block membranes in 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary CCDC153 antibody at 1:500-1:2000 dilution (optimize for each antibody)

  • For COLO 320 cell lysates, a 1:300 dilution has been specifically validated

• Incubate overnight at 4°C or 1-2 hours at room temperature

• Wash 3-5 times with TBST

• Incubate with appropriate HRP-conjugated secondary antibody (anti-rabbit IgG)

• Develop using chemiluminescence detection

Expected results:

• The predominant band should appear at 45-48 kDa (dimeric form)

• A fainter band at 24 kDa may be visible in some samples (monomeric form)

• Validated positive controls include: COLO 320 cells, HepG2 cells, L02 cells, human brain tissue, and HeLa cells

For troubleshooting weak or absent signals, consider increasing antibody concentration, extending incubation time, or using enhanced sensitivity detection reagents. For non-specific bands, more stringent washing or higher dilution of primary antibody may be beneficial.

How should immunofluorescence experiments with CCDC153 antibody be optimized for different cell types?

Immunofluorescence (IF) detection of CCDC153 requires careful optimization to achieve specific labeling with minimal background. Based on validated protocols, the following approach is recommended for various cell types:

General IF protocol:

• Culture cells on coverslips or chamber slides to 70-80% confluence

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes

• Block with 1-5% BSA or normal serum in PBS for 30-60 minutes

• Incubate with CCDC153 primary antibody at the appropriate dilution

• Incubate with fluorophore-conjugated secondary antibody (e.g., Rhodamine-labeled goat anti-rabbit IgG)

• Counterstain nuclei with DAPI or other DNA dye

• Mount and visualize using fluorescence microscopy

Cell type-specific considerations:

For HepG2 cells specifically, immunofluorescence analysis using CCDC153 antibody at 1:25 dilution with Rhodamine-labeled goat anti-rabbit IgG secondary antibody has been validated to produce clear, specific signals . DAPI counterstaining allows for visualization of nuclear morphology and assessment of subcellular localization of CCDC153.

When working with new cell types, titration experiments starting within the recommended dilution range (1:10-1:100) are essential to determine optimal antibody concentration . Signal-to-noise ratio should be the primary criterion for selecting the optimal dilution.

How does CCDC153 antibody performance compare across different immunohistochemistry platforms and detection systems?

The performance of CCDC153 antibody in immunohistochemistry (IHC) can vary significantly depending on the detection system and platform used. Understanding these differences is crucial for selecting the optimal approach for specific research questions.

Comparison across common IHC detection systems:

Tissue-specific considerations:

• Human prostate hyperplasia tissue has been specifically validated for CCDC153 IHC

• Recommended dilution range is 1:20-1:200, with 1:50 specifically validated for prostate tissue

• Antigen retrieval using TE buffer pH 9.0 is the primary recommendation, with citrate buffer pH 6.0 as an alternative

Platform-specific optimization:

• Manual IHC:

  • Provides greatest flexibility for protocol optimization

  • Critical steps include adequate blocking and careful antibody titration

  • Validated on paraffin-embedded tissues at 1:50 dilution

• Automated platforms:

  • May require adaptation of incubation times and antibody concentrations

  • Often benefit from platform-specific detection kits

  • Generally provide more consistent results across multiple slides

• Whole slide imaging:

  • Compatible with CCDC153 IHC staining

  • Useful for quantitative analysis of staining patterns and intensity

For all platforms, proper controls are essential, including positive controls (human prostate hyperplasia tissue) and negative controls (primary antibody omission or isotype controls). Due to the relatively limited characterization of CCDC153, extensive validation is particularly important when implementing new detection systems or platforms.

What is currently known about CCDC153's biological function and its potential as a research target?

• Neuronal subtype marker: Research indicates that CCDC153 may serve as a marker for specific neuronal subtypes (PMID: 28166221) . This suggests potential applications in neuroscience research, particularly in studies examining neuronal differentiation, classification, or circuit mapping.

• Coiled-coil domain functionality: As a member of the coiled-coil domain containing protein family, CCDC153 likely participates in protein-protein interactions, potentially forming homo- or heterodimers with other proteins. The observation that it frequently appears as a dimer (45-48 kDa) on Western blots supports this functional characteristic .

• Tissue expression pattern: Current validation data show CCDC153 expression in various cell types including COLO 320 cells (colorectal adenocarcinoma), HepG2 cells (hepatocellular carcinoma), L02 cells (normal hepatic), human brain tissue, and HeLa cells (cervical cancer) . This diverse expression pattern suggests potential roles in multiple tissue types.

• Subcellular localization: Immunofluorescence studies in HepG2 and HeLa cells could provide insights into the protein's subcellular localization , though specific details about its distribution pattern are not provided in the available search results.

Despite these initial insights, significant knowledge gaps remain regarding CCDC153's:

• Specific molecular interactions and binding partners

• Regulation of expression under different physiological or pathological conditions

• Potential involvement in disease processes

• Evolutionary conservation and structural features

These knowledge gaps present opportunities for future research using CCDC153 antibodies for techniques such as co-immunoprecipitation, proximity ligation assays, or ChIP-seq to elucidate the protein's functional networks and regulatory mechanisms.

How can researchers troubleshoot common issues when using CCDC153 antibody in experimental applications?

When working with CCDC153 antibody, researchers may encounter various technical challenges. The following troubleshooting guide addresses common issues across different applications:

Western Blot Troubleshooting:

Immunohistochemistry Troubleshooting:

Immunofluorescence Troubleshooting:

General Recommendations:

• Always include positive controls (e.g., COLO 320 cells, HepG2 cells, human brain tissue)

• Implement proper negative controls (isotype control, primary antibody omission)

• For new applications or cell types, perform antibody titration experiments

• Consider batch effects of antibody lots when comparing experiments over time

By systematically addressing these common issues using the recommended approaches, researchers can optimize CCDC153 antibody performance across experimental applications.

What experimental design considerations are important when conducting co-localization studies with CCDC153 antibody?

Co-localization studies with CCDC153 antibody require careful experimental design to generate reliable and interpretable results. The following considerations should guide researchers planning such experiments:

Antibody selection and validation:

• Ensure CCDC153 rabbit polyclonal antibody is compatible with other primary antibodies (ideally from different host species)

• Validate antibody specificity in the specific cell type or tissue being studied

• For HepG2 and HeLa cells, CCDC153 antibody has been validated for immunofluorescence applications

Microscopy and imaging considerations:

• Selection of appropriate microscopy platform:

  • Confocal microscopy is preferred for definitive co-localization studies

  • Super-resolution techniques (STED, STORM, etc.) may be necessary for detailed subcellular localization

• Fluorophore selection:

  • Use spectrally distinct fluorophores to avoid bleed-through

  • Rhodamine-labeled goat anti-rabbit IgG has been validated as a secondary antibody

  • DAPI counterstaining for nuclear visualization works well with CCDC153 immunofluorescence

• Controls for co-localization:

  • Positive control: Co-stain with markers known to co-localize

  • Negative control: Co-stain with markers of distinct subcellular compartments

  • Single-label controls to rule out bleed-through artifacts

Protocol optimization:

• Fixation and permeabilization:

  • Standard 4% paraformaldehyde fixation is typically sufficient

  • Optimize Triton X-100 concentration (0.1-0.5%) for balanced permeabilization

• Antibody dilution:

  • For CCDC153: 1:10-1:100 range is recommended for IF

  • 1:25 dilution specifically validated for HepG2 cells

  • Titrate co-staining antibodies independently before combining

• Sequential vs. simultaneous incubation:

  • If antibodies are from different host species, simultaneous incubation is possible

  • For same-host antibodies, sequential protocols with intermediate blocking steps are required

Quantitative analysis:

• Apply appropriate co-localization metrics (Pearson's correlation, Manders' coefficients)

• Use software tools that correct for background and threshold appropriately

• Perform statistical analysis across multiple cells and experimental replicates

Given the limited characterization of CCDC153, co-localization studies could provide valuable insights into its subcellular distribution and potential functional interactions. Validated cell models such as HepG2 and HeLa cells provide a solid starting point for such investigations.

What are the current best practices for utilizing CCDC153 antibody in multi-omics and integrative research approaches?

CCDC153 antibody can be effectively integrated into multi-omics and integrative research approaches to advance understanding of this relatively uncharacterized protein. Best practices include:

• Cross-validation across platforms:

  • Confirm CCDC153 expression using multiple techniques (Western blot, IHC, IF)

  • Validated cell lines such as COLO 320, HepG2, and HeLa provide reliable experimental systems

  • Compare antibody-based protein detection with transcriptomics data for concordance

• Integration with proteomics approaches:

  • Use CCDC153 antibody for immunoprecipitation followed by mass spectrometry

  • Validate the 45-48 kDa dimeric form observed in Western blots

  • Identify interaction partners to build functional protein networks

• Combination with genomics and transcriptomics:

  • Correlate protein expression (antibody-based) with RNA expression

  • Investigate epigenetic regulation of CCDC153 in different cell types

  • Consider genetic variants that may affect antibody epitope recognition

• Spatial biology applications:

  • Apply validated IHC protocols (1:20-1:200 dilution) for tissue microarrays

  • Combine with single-cell transcriptomics to correlate protein localization with gene expression

  • Consider multiplexed IHC/IF for contextual cellular information

• Functional genomics integration:

  • Use CCDC153 antibody to validate CRISPR/siRNA knockdown efficiency

  • Compare phenotypes with protein expression levels across experimental conditions

  • Apply in combination with live-cell imaging or reporter systems

Each of these approaches should incorporate rigorous controls and validation steps, particularly given the relatively limited characterization of CCDC153. Researchers should also consider the molecular weight discrepancy (calculated 24 kDa vs. observed 45-48 kDa) when interpreting results across different platforms.