CCDC158 Antibody

What is CCDC158 and what are its primary biological functions?

CCDC158 is a protein characterized by coiled-coil domains with a calculated molecular weight of 127 kDa (1113 amino acids), though it typically appears as 127-140 kDa bands in Western blots. Recent research has identified CCDC158 as a novel regulator in renal proximal tubular endocytosis . The protein appears to play a critical role in receptor-mediated endocytosis, particularly in the kidney, where it participates in the reabsorption of proteins and polypeptides through interaction with multi-ligand receptors and endocytic pathway components . This process involves progression from clathrin-coated pits to early/recycling endosomes and ultimately to late endosomes and lysosomes.

What tissues express CCDC158 and how is this relevant to research applications?

CCDC158 demonstrates notable tissue-specific expression patterns:

The expression in kidney and testis tissues correlates with clinical findings where CCDC158 variants have been associated with both renal dysfunction (proteinuria and nephrocalcinosis) and reproductive issues (oligospermia) in male patients . These expression patterns guide researchers in selecting appropriate positive control tissues and understanding potential phenotypes in functional studies.

What are the validated applications for CCDC158 antibody?

The rabbit polyclonal CCDC158 antibody has been validated for multiple research applications:

Researchers should note that optimal dilutions may be sample-dependent and should be determined empirically for each experimental system .

What are the optimal conditions for immunohistochemistry with CCDC158 antibody?

For optimal IHC results with CCDC158 antibody:

• Antigen retrieval recommendation: TE buffer pH 9.0 is suggested as the primary method

• Alternative method: Citrate buffer pH 6.0 can also be used as an alternative

• Dilution range: 1:20-1:200, with optimization recommended for each tissue type

• Positive control tissues: Human kidney and testis tissues have been validated

• Detection system: Compatible with standard detection methods

For paraffin-embedded tissues, proper antigen retrieval is critical for exposing the epitopes recognized by the antibody. The difference in results between pH 9.0 and pH 6.0 buffers may reflect the structural characteristics of the CCDC158 protein and its preservation in fixed tissues.

How should the CCDC158 antibody be stored and handled to maintain optimal activity?

Proper storage and handling are essential for maintaining antibody performance:

To preserve antibody activity, avoid repeated freeze-thaw cycles and centrifuge the vial briefly before use to ensure the solution is at the bottom of the container .

How can researchers validate the specificity of CCDC158 antibody in their experimental system?

Validation of antibody specificity is crucial for accurate data interpretation. For CCDC158 antibody, consider these validation strategies:

• Western blot validation:

  • Compare observed band size (127-140 kDa) with predicted molecular weight

  • Use known positive tissues (mouse or human testis) as controls

  • Consider knockdown experiments to confirm specificity

• IHC validation:

  • Include appropriate positive control tissues (human kidney, testis)

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

  • Compare staining patterns with published results

• Cross-validation approaches:

  • Confirm protein expression using multiple techniques (WB, IHC, IF)

  • Correlate protein detection with mRNA expression data

  • Consider mass spectrometry confirmation for IP experiments

What is known about CCDC158's role in renal proximal tubular endocytosis?

CCDC158 has been identified as an important regulator in renal proximal tubular endocytosis based on recent research. Whole exome-sequencing of patients from a consanguineous family revealed that variants in CCDC158 correlate with low- and intermediate molecular weight proteinuria and nephrocalcinosis . The protein appears to function in the highly regulated process of protein reabsorption in the proximal tubule through:

• Interaction with the endocytic machinery, particularly with clathrin-coated structures

• Facilitation of trafficking between early/recycling endosomes and late endosomes

• Participation in the lysosomal degradation pathway for reabsorbed proteins

Mass spectrometry analysis has identified potential protein interactors, suggesting CCDC158 functions within a complex network of proteins involved in endocytosis regulation . Transferrin and GST-RAP trafficking analyses in patient-derived proximal tubular epithelial cells further confirmed CCDC158's role in receptor-mediated endocytosis.

How does CCDC158 dysfunction manifest in pathological conditions?

Research has linked CCDC158 variants to specific pathological conditions:

The renal phenotype strongly correlates with the proposed function of CCDC158 in tubular endocytosis, as defective protein reabsorption leads to increased urinary protein excretion . The reproductive phenotype aligns with CCDC158's expression in testicular tissue, though the specific mechanism remains to be fully elucidated.

What troubleshooting approaches are recommended for weak or non-specific signals?

When encountering difficulties with CCDC158 antibody performance:

For Western Blot issues:

• Increase primary antibody concentration (try 1:500 instead of 1:2000)

• Extend primary antibody incubation (overnight at 4°C)

• Optimize protein loading (50-100 μg total protein recommended)

• Test different blocking reagents (5% non-fat milk vs. BSA)

• Consider fresh lysates to avoid protein degradation

For IHC challenges:

• Compare antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Adjust antibody concentration (starting with 1:20 for weak signals)

• Extend primary antibody incubation time

• Verify tissue fixation quality (over/under-fixation can affect epitope accessibility)

General considerations:

• Verify antibody within shelf-life and proper storage conditions

• Include established positive controls in every experiment

• Consider tissue-specific expression levels of CCDC158

How does CCDC158 interact with other proteins in endocytic pathways?

Research suggests CCDC158 functions within a complex protein network involved in endocytosis regulation. The coiled-coil domains characteristic of this protein likely facilitate protein-protein interactions essential for endocytic functions . Mass spectrometry studies have identified potential interacting partners, though complete characterization of these interactions requires further research.

The interaction between CCDC158 and components of clathrin-coated structures appears particularly important, suggesting a role in the early stages of endocytosis. Additionally, CCDC158 may participate in sorting mechanisms that direct internalized proteins to either recycling or degradation pathways.

What research models are available for studying CCDC158 function?

Several experimental systems have been employed to study CCDC158 function:

• Patient-derived proximal tubular epithelial cells: Valuable for studying endocytic defects in cells carrying CCDC158 variants

• Transferrin and GST-RAP trafficking assays: Useful for measuring receptor-mediated endocytosis efficiency

• Structural modeling: In silico approaches have been used to model CCDC158 structure and predict functional domains

Future research might benefit from developing animal models with CCDC158 mutations to further characterize its physiological roles in vivo. Cell culture systems expressing fluorescently tagged CCDC158 could also provide insights into its subcellular localization and trafficking dynamics.

What are the future research directions for understanding CCDC158 function?

While significant progress has been made in identifying CCDC158 as a regulator of renal endocytosis, several areas require further investigation:

• Complete characterization of CCDC158's molecular partners and protein interaction network

• Detailed mapping of functional domains within the protein structure

• Elucidation of signaling pathways that regulate CCDC158 expression and activity

• Investigation of potential therapeutic approaches targeting CCDC158 pathways for treating related kidney disorders

• Understanding the role of CCDC158 in reproductive biology and male fertility

As noted by researchers, "more studies are needed to fully unravel the molecular mechanism(s) in which CCDC158 is involved" . The continued development and utilization of specific antibodies against CCDC158 will be crucial for advancing these research goals.

What are the detailed specifications of commercially available CCDC158 antibodies?

This rabbit polyclonal antibody recognizes both human and mouse CCDC158 proteins, with predicted reactivity to rat samples as well . The antibody's polyclonal nature provides recognition of multiple epitopes, which can be advantageous for applications like IHC where protein conformation may be altered during fixation.

How do different experimental protocols affect CCDC158 antibody performance?

The performance of CCDC158 antibody varies across different experimental protocols:

Western Blot considerations:

• Reducing conditions are compatible with epitope detection

• Transfer efficiency is critical due to the large size of the protein (127-140 kDa)

• Extended transfer times may be necessary for complete protein migration

IHC protocol factors:

• Formalin-fixed paraffin-embedded tissues require appropriate antigen retrieval

• TE buffer pH 9.0 is recommended for optimal epitope exposure

• Citrate buffer pH 6.0 provides an alternative with potentially different epitope exposure

IP protocol optimization:

• 0.5-4.0 μg antibody is recommended for immunoprecipitating CCDC158 from 1.0-3.0 mg of total protein lysate

• Pre-clearing lysates may reduce non-specific binding

• Appropriate controls are essential for interpreting results