PRKCDBP Antibody

What is PRKCDBP and what are its known functions?

PRKCDBP (Protein Kinase C Delta Binding Protein), also known as cavin-3, belongs to the cavin family of proteins involved in caveolin formation and regulation. It was initially identified in a screen of cultured cell lines as a protein strongly induced by serum starvation . PRKCDBP binds to PKC-δ and caveolin-1, helping regulate caveolin trafficking and function . Additionally, it is a BRCA1-interacting protein that may participate in DNA damage response pathways . As a putative tumor suppressor, alterations in PRKCDBP have been observed in several human cancers, including ovarian, gastric, and lung cancers .

What is the subcellular localization of PRKCDBP?

Based on immunofluorescence studies in MCF-7 cells and other cell lines, PRKCDBP primarily localizes to the plasma membrane, particularly in caveolae structures, consistent with its role in caveolin trafficking and regulation . Additionally, some nuclear localization has been reported, which may relate to its potential role in DNA damage response pathways through BRCA1 interaction.

What types of PRKCDBP antibodies are available for research?

There are two main types of PRKCDBP antibodies available for research:

Polyclonal antibodies typically recognize multiple epitopes and may provide higher sensitivity, while monoclonal antibodies offer greater specificity for a single epitope .

How should I validate a PRKCDBP antibody for my research?

Validation of PRKCDBP antibodies should follow these methodological steps:

• Western blot validation: Confirm antibody detects a band of the expected molecular weight (35-42 kDa) in appropriate positive control samples (e.g., mouse lung tissue, MCF-7 cells, HeLa cells) .

• Immunoprecipitation validation: Verify antibody can pull down PRKCDBP from tissue or cell lysates (e.g., mouse lung tissue, A549 cells) .

• Specificity testing: Use knockout/knockdown models or blocking peptides to confirm specificity .

• Cross-reactivity assessment: Test antibody against recombinant PRKCDBP protein and related family members to ensure minimal cross-reactivity .

• Application-specific validation: Validate for each specific application (WB, IHC, IF) using recommended dilutions and positive control samples .

What are recommended positive control samples for PRKCDBP antibody validation?

Based on the search results, the following samples are recommended as positive controls for PRKCDBP antibody validation:

• Tissues: Human lung cancer tissue, normal human colon, mouse lung tissue

• Cell lines: MCF-7 cells, HeLa cells, A549 cells

• Recombinant proteins: GST-tagged PRKCDBP recombinant protein

When validating antibodies in Western blot applications, expect to observe a band at approximately 35-42 kDa in positive control samples .

What are the recommended dilutions for different applications of PRKCDBP antibodies?

Optimal antibody dilutions vary by application and specific antibody. Based on the search results, these general guidelines can serve as starting points:

Always validate these dilutions for your specific experimental conditions, as optimal concentrations may vary by lot and sample type .

What is the recommended protocol for immunohistochemical detection of PRKCDBP?

For successful immunohistochemical detection of PRKCDBP in tissue samples:

• Tissue preparation: Use formalin-fixed paraffin-embedded (FFPE) tissue sections .

• Antigen retrieval: Some antibodies may require antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0) .

• Blocking: Block non-specific binding using appropriate serum or protein blockers.

• Primary antibody incubation: Apply PRKCDBP antibody at recommended dilution (typically 1:50-1:500 or 5 μg/mL) and incubate at appropriate temperature and duration (e.g., overnight at 4°C) .

• Detection system: Use appropriate detection system (e.g., HRP-conjugated secondary antibody) and visualize with DAB or similar substrate.

• Controls: Include positive control tissues (e.g., human lung tissue) and negative controls (primary antibody omission) .

PRKCDBP has been successfully detected in human lung cancer tissue and normal colon tissue using this approach .

What is the optimal protocol for Western blot detection of PRKCDBP?

For optimal Western blot detection of PRKCDBP:

• Sample preparation: Prepare lysates from appropriate tissues (e.g., lung tissue) or cell lines (e.g., MCF-7, HeLa cells) .

• Protein loading: Load 20-50 μg of total protein per lane.

• Gel separation: Use 10-12% SDS-PAGE gels for optimal separation around the 35-42 kDa range.

• Transfer: Transfer to PVDF or nitrocellulose membrane using standard protocols.

• Blocking: Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Incubate with anti-PRKCDBP antibody at 1:200-1:1000 dilution overnight at 4°C .

• Secondary antibody: Use appropriate HRP-conjugated secondary antibody.

• Detection: Visualize using enhanced chemiluminescence (ECL).

• Expected results: Look for a band at approximately 35-42 kDa, which is the observed molecular weight for PRKCDBP despite its calculated weight of ~28 kDa .

Why is there a discrepancy between calculated and observed molecular weights for PRKCDBP?

The calculated molecular weight of PRKCDBP is approximately 27-30 kDa based on amino acid sequence, but it typically appears at 35-42 kDa in Western blot analyses . This discrepancy may be attributed to:

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications can increase apparent molecular weight .

• Protein structure: Tertiary structure or high proline content can affect migration in SDS-PAGE.

• Isoforms: Alternatively spliced variants may exist with different molecular weights.

• Technical factors: Buffer conditions, gel percentage, and running conditions can affect protein migration.

This discrepancy is consistently reported across multiple sources and should be expected when analyzing experimental results .

What are common issues when detecting PRKCDBP in immunohistochemistry and how can I resolve them?

Common issues and solutions for PRKCDBP immunohistochemistry include:

When optimizing, always include positive controls (e.g., human lung cancer tissue) for comparison .

How can I interpret PRKCDBP expression patterns in cancer tissues?

When interpreting PRKCDBP expression in cancer tissues:

• Expression patterns: PRKCDBP typically shows membrane and cytoplasmic localization, with possible nuclear staining in some cases .

• Expression in cancers: As a putative tumor suppressor, PRKCDBP may show decreased expression in certain cancers compared to normal tissues. Altered expression has been observed in lung, ovarian, and gastric cancers .

• Correlation with clinical data: Consider correlating expression levels with clinical parameters (stage, grade, patient outcomes) for comprehensive analysis.

• Quantification: Use appropriate scoring systems (e.g., H-score, percentage positive cells) for semi-quantitative analysis.

• Controls: Always compare with normal tissue controls and validate findings with alternative methods (e.g., qPCR, Western blot) .

When interpreting results, remember that antibody specificity is crucial, and validation with multiple detection methods is recommended for conclusive findings .

How can PRKCDBP antibodies be used to study its tumor suppressor function?

To investigate PRKCDBP's tumor suppressor function:

• Expression analysis: Compare PRKCDBP expression between normal and tumor tissues using validated antibodies in IHC and Western blot analyses .

• Correlation studies: Analyze relationships between PRKCDBP expression levels and clinical parameters (tumor stage, grade, patient survival) .

• Mechanism studies: Use PRKCDBP antibodies in co-immunoprecipitation experiments to identify interaction partners (e.g., BRCA1, caveolin-1, PKC-δ) and elucidate molecular mechanisms .

• Functional studies: Combine antibody-based detection methods with PRKCDBP knockdown/overexpression models to correlate protein levels with phenotypic changes.

• DNA damage response: Investigate PRKCDBP localization and expression changes following DNA damage induction, particularly in relation to its interaction with BRCA1 .

This multi-faceted approach can provide insights into how alterations in PRKCDBP contribute to cancer development and progression .

How can I use PRKCDBP antibodies to study its interaction with caveolin and PKC-δ?

To investigate PRKCDBP interactions with caveolin and PKC-δ:

• Co-immunoprecipitation: Use PRKCDBP antibodies to pull down protein complexes, then probe for caveolin-1 and PKC-δ by Western blot .

• Reverse co-IP: Immunoprecipitate with anti-caveolin-1 or anti-PKC-δ antibodies and probe for PRKCDBP.

• Proximity ligation assay (PLA): Visualize protein-protein interactions in situ using PRKCDBP antibodies in combination with caveolin-1 or PKC-δ antibodies.

• Co-localization studies: Perform dual immunofluorescence staining with PRKCDBP and caveolin-1 or PKC-δ antibodies to assess subcellular co-localization .

• Stimulus-dependent interactions: Examine how these interactions change under different conditions (e.g., serum starvation, which was reported to induce PRKCDBP expression) .

These approaches can help elucidate the molecular mechanisms by which PRKCDBP regulates caveolin trafficking and function, and its role in PKC-δ signaling pathways .

What methodological approaches can be used to study PRKCDBP in DNA damage response pathways?

To investigate PRKCDBP's role in DNA damage response pathways:

• DNA damage induction: Treat cells with DNA-damaging agents (e.g., ionizing radiation, etoposide) and analyze PRKCDBP expression and localization changes using validated antibodies .

• Co-localization with DNA damage markers: Perform co-immunofluorescence for PRKCDBP and DNA damage markers (γ-H2AX, 53BP1) to assess recruitment to damage sites.

• BRCA1 interaction studies: Use co-immunoprecipitation with PRKCDBP antibodies to examine interactions with BRCA1 under normal and DNA damage conditions .

• Functional studies: Combine PRKCDBP knockdown/overexpression with DNA damage assays (comet assay, HR/NHEJ reporter assays) and monitor repair efficiency.

• Chromatin immunoprecipitation (ChIP): Assess whether PRKCDBP is recruited to chromatin at DNA damage sites.

• Cell cycle analysis: Examine how PRKCDBP expression and localization changes throughout the cell cycle, particularly following DNA damage checkpoint activation.

These methodological approaches can help elucidate the potential role of PRKCDBP in the DNA damage response pathway and its functional relationship with BRCA1 .