PDZK1 Antibody

What is PDZK1 and why is it important in cellular research?

PDZK1 is a scaffold protein that connects plasma membrane proteins and regulatory components, playing a critical role in regulating their surface expression in epithelial cells' apical domains. It coordinates diverse regulatory processes for ion transport and second messenger cascades, functioning in complex with SLC9A3R1 to cluster functionally dependent proteins and modulate the trafficking and activity of associated membrane proteins. Its significance extends to cellular mechanisms of multidrug resistance through interactions with ABCC2 and PDZK1IP1, and it may potentiate CFTR chloride channel activity . In recent studies, PDZK1 has emerged as a significant prognostic factor for triple-negative breast cancer (TNBC) and a potential molecular therapeutic target for reversing erlotinib resistance in TNBC cells .

What types of PDZK1 antibodies are available and how should I select one for my research?

Multiple types of PDZK1 antibodies are available, varying by host species, clonality, and epitope recognition. The available options include:

• Polyclonal antibodies: Generally from rabbit hosts, recognizing multiple epitopes across the PDZK1 protein, useful for robust detection of low-abundance proteins

• Monoclonal antibodies: Available from mouse or rabbit hosts, targeting specific epitopes with high specificity, ideal for distinguishing between closely related proteins

• Species reactivity profiles: Antibodies with human-only specificity or broader cross-reactivity with mouse, rat, and other species

Selection should be based on your experimental application (WB, IHC, IF), required specificity, and target species. For example, sheep anti-human/mouse PDZK1 antibodies have been successfully used in Western blot analysis of human cancer cell lines and mouse liver tissue, showing a specific band at approximately 80 kDa .

What are the validated applications for PDZK1 antibodies in research protocols?

PDZK1 antibodies have been validated for multiple experimental applications, each requiring specific optimization:

For optimal results in Western blotting, researchers should probe PVDF membranes with specific antibody concentrations (e.g., 1 μg/mL of sheep anti-human/mouse PDZK1 antibody), followed by appropriate HRP-conjugated secondary antibodies like anti-sheep IgG .

How should samples be prepared for optimal PDZK1 detection in Western blotting?

Sample preparation significantly impacts PDZK1 detection quality. Based on established protocols:

• Cell lysis should be performed using buffers containing appropriate detergents (e.g., TBS with protease inhibitors including 1 μM pepstatin, 1 μM leupeptin, and 230 μM PMSF)

• Solubilization of membrane proteins is critical - 1.25 mg of membrane preparations (e.g., brush border membrane vesicles) can be solubilized in 5 ml of TBS solubilization buffer

• For reduced conditions, include β-mercaptoethanol or DTT in loading buffer

• Run protein samples on 10-20% polyacrylamide gradient gels for optimal resolution

• Transfer to PVDF membrane is preferred over nitrocellulose for PDZK1 detection

• Blocking should be performed using 5% nonfat dried milk in TBS/0.1% Tween-20 with protease inhibitors for 4 hours at 4°C

Following these preparation steps ensures reproducible detection of the ~80 kDa PDZK1 protein across various sample types including human cancer cell lines and tissue lysates .

What are the recommended protocols for immunohistochemical detection of PDZK1?

Immunohistochemical detection of PDZK1 requires specific optimization steps:

• Tissue fixation: Immersion fixation in formaldehyde followed by paraffin embedding preserves PDZK1 epitopes

• Antigen retrieval: Heat-induced epitope retrieval may be necessary depending on fixation method

• Primary antibody concentration: 15 μg/mL has been validated for sheep anti-human/mouse PDZK1 antibody

• Incubation conditions: Overnight incubation at 4°C provides optimal signal-to-noise ratio

• Detection system: For DAB visualization, anti-sheep HRP-DAB staining kits have been successfully employed

• Counterstaining: Hematoxylin provides good contrast for visualizing PDZK1-positive structures

In human kidney sections, this approach results in specific labeling localized to the brush border of proximal tubules, consistent with PDZK1's known biological distribution .

How can PDZK1 antibodies be used to investigate protein-protein interactions?

PDZK1 functions through multiple protein-protein interactions that can be studied using antibody-based approaches:

• Co-immunoprecipitation: PDZK1 antibodies can precipitate protein complexes containing PDZK1 and its binding partners. This technique has revealed interactions with proteins such as EGFR, demonstrating that PDZK1 promotes EGFR degradation by enhancing binding of EGFR to c-Cbl .

• GST-pulldown assays: GST-PDZK1 fusion proteins can be used in overlay experiments to identify direct interactions. This approach has demonstrated that PDZK1 interacts with the C-terminal PDZ-interaction motif of proteins like NHE3 .

• Proximity ligation assay: This advanced technique can visualize protein interactions in situ with high specificity using pairs of antibodies and has been informative in studying PDZK1 complexes.

• Immunofluorescence co-localization: Double-labeling with PDZK1 antibodies and antibodies against potential interaction partners can provide spatial information about protein associations.

The specificity of these interaction studies should be validated using approaches such as peptide competition or comparison with characterized antibodies like the chicken anti-human PDZK1 antibody .

What role does PDZK1 play in cancer biology and how can antibodies help investigate this function?

Recent research has revealed significant implications of PDZK1 in cancer biology, particularly in TNBC:

• PDZK1 is specifically downregulated in TNBC tissues compared to normal tissues and correlates with poor prognosis in TNBC patients .

• PDZK1 expression levels negatively correlate with EGFR pathway activation in TNBC samples, as demonstrated by gene set enrichment analysis (GSEA) and immunohistochemistry on tissue microarrays .

• PDZK1 suppresses TNBC development by:

  • Binding to EGFR and promoting its degradation

  • Enhancing EGFR binding to c-Cbl

  • Inhibiting EGFR phosphorylation by hindering EGFR dimerization

• PDZK1 levels correlate with erlotinib sensitivity in TNBC cells:

  • PDZK1 is downregulated in erlotinib-resistant TNBC cells (MDA-MB-231, MDA-MB-468)

  • Higher PDZK1 expression is observed in erlotinib-sensitive/moderately sensitive cells (SUM149, SK-BR-3)

PDZK1 antibodies are crucial tools for investigating these mechanisms through techniques such as Western blotting to quantify expression levels, immunohistochemistry to assess tissue localization patterns, and co-immunoprecipitation to study EGFR-PDZK1 interactions .

How can researchers troubleshoot non-specific binding issues with PDZK1 antibodies?

Non-specific binding can complicate PDZK1 detection. Systematic troubleshooting approaches include:

• Antibody validation:

  • Peptide blocking experiments using the immunizing peptide (e.g., the 29-aa peptide CEKDTEGHLVRVVEQGSPAEKAGLKDGDR used for OK-66 antibody production)

  • Comparison with previously characterized antibodies

  • Testing in PDZK1 knockout or knockdown samples

• Optimization of blocking conditions:

  • Extended blocking (4+ hours at 4°C) with 5% nonfat dried milk in TBS/0.1% Tween-20 with protease inhibitors

  • Alternative blocking agents like BSA or commercial blocking solutions if milk proves inadequate

• Stringent washing protocols:

  • Multiple washes (6 or more) of 10 minutes each with cold blocking solution

  • Increased detergent concentration in wash buffers

• Dilution optimization:

  • Titrating antibody concentrations (e.g., testing dilutions from 1:1,000 to 1:10,000)

  • Using affinity-purified antibody preparations

• Cross-reactivity assessment:

  • Testing against multiple species to confirm expected reactivity patterns

  • Note that while some antibodies (like OK-66) recognize PDZK1 across species, others may have limitations

How does PDZK1 expression correlate with EGFR-targeted therapy resistance?

PDZK1 has emerged as a key factor in EGFR-targeted therapy resistance, particularly for erlotinib treatment in TNBC:

• Expression correlation:

  • PDZK1 is downregulated in erlotinib-resistant TNBC cell lines (MDA-MB-231, MDA-MB-468) compared to erlotinib-sensitive/moderately sensitive lines (SUM149, SK-BR-3)

  • Low PDZK1 expression is associated with EGFR pathway activation in TNBC samples as determined by GSEA and immunohistochemistry

• Mechanistic insights:

  • PDZK1 binds directly to EGFR

  • Through this interaction, PDZK1 promotes EGFR degradation by enhancing EGFR binding to c-Cbl

  • PDZK1 inhibits EGFR phosphorylation by preventing EGFR dimerization

• Clinical significance:

  • PDZK1 downregulation correlates with poor prognosis specifically in TNBC patients

  • This correlation is not detected in non-TNBC patients, suggesting TNBC-specific effects

These findings position PDZK1 as a potential molecular therapeutic target for reversing erlotinib resistance in TNBC cells and as a significant prognostic factor for TNBC .

What methodologies are recommended for studying PDZK1 in different tissue contexts?

Different tissues require specific methodological approaches for optimal PDZK1 detection and characterization:

• Kidney tissue:

  • Immunohistochemistry using 15 μg/mL antibody concentration on paraffin-embedded sections

  • Overnight incubation at 4°C followed by HRP-DAB visualization

  • Specific labeling is expected at the brush border of proximal tubules

• Cancer cell lines:

  • Western blotting using 1 μg/mL antibody concentration for various cancer cell lines (e.g., Calu-6, U266, MCF-7)

  • PVDF membrane with reducing conditions and appropriate buffer systems

  • Expected band at approximately 80 kDa

• Liver tissue:

  • Western blotting can effectively detect PDZK1 in mouse liver tissue

  • Gene expression analysis through qPCR to quantify transcriptional changes

• Triple-negative breast cancer:

  • Tissue microarray analysis with immunohistochemistry to correlate PDZK1 levels with clinical outcomes

  • Gene set enrichment analysis to investigate pathway associations

  • In vitro and in vivo functional assays using PDZK1 overexpression or knockdown to assess effects on cancer development

• Cross-species investigation:

  • The OK-66 antibody has been validated to recognize PDZK1 across multiple species including human, mouse, rat, rabbit, and opossum

  • Degenerate PCR approaches based on known sequences can be used to isolate PDZK1 domains from different species

How can researchers quantitatively assess PDZK1 expression in clinical samples?

Quantitative assessment of PDZK1 in clinical contexts requires standardized approaches:

• Immunohistochemical scoring systems:

  • Semi-quantitative scoring considering staining intensity (0-3) and percentage of positive cells

  • Digital image analysis for objective quantification of staining patterns

  • Correlation with clinical parameters including patient survival

• Western blot quantification:

  • Densitometric analysis normalized to loading controls

  • Standard curves using recombinant PDZK1 for absolute quantification

  • Comparison across patient cohorts stratified by disease characteristics

• mRNA expression analysis:

  • qRT-PCR with appropriate reference genes

  • RNA-seq for genome-wide expression context

  • Correlation between protein and mRNA levels to identify post-transcriptional regulation

• Tissue microarray technology:

  • Enables high-throughput analysis across large patient cohorts

  • Statistical correlation with clinical parameters

  • Multivariate analysis to identify independent prognostic value

These quantitative approaches have revealed that PDZK1 is specifically downregulated in TNBC tissues compared to normal tissues and correlates with poor prognosis specifically in TNBC patients but not in non-TNBC patients .

What are the emerging applications of PDZK1 antibodies in precision medicine?

PDZK1's role in erlotinib sensitivity suggests several emerging applications:

• Predictive biomarker development:

  • PDZK1 immunohistochemistry could potentially predict TNBC patient response to EGFR-targeted therapies like erlotinib

  • Standardized scoring systems need development for clinical implementation

• Therapeutic targeting strategies:

  • Screening for compounds that stabilize PDZK1 or prevent its degradation

  • Development of mimetic peptides that reproduce PDZK1's interaction with EGFR

  • Therapeutic antibodies targeting pathways that regulate PDZK1 expression

• Combination therapy rationales:

  • Using PDZK1 status to guide combination approaches with erlotinib

  • Targeting parallel pathways in PDZK1-low tumors

• Monitoring treatment response:

  • Serial assessment of PDZK1 levels during therapy

  • Correlation with clinical outcomes and resistance development

Recent research demonstrating that PDZK1 suppresses TNBC development and sensitizes TNBC cells to erlotinib establishes this protein as both a prognostic factor and potential therapeutic target, opening new avenues for precision oncology approaches .