PRKD1 Antibody, Biotin conjugated

What is PRKD1 and why is it significant in cellular research?

PRKD1 (Protein Kinase D1) is a serine/threonine protein kinase with a calculated molecular weight of 102 kDa and plays crucial roles in cellular signaling pathways. The significance of PRKD1 lies in its involvement in regulating cell proliferation, motility, and invasion processes, particularly in cancer progression. Recent studies have demonstrated that PRKD1 functions as a potential tumor suppressor, as its expression is significantly downregulated in invasive breast cancer tissues compared to normal breast epithelium . The gene is encoded by the PRKD1 gene (GenBank Accession Number NM_002742), and the protein has been assigned UNIPROT ID Q15139 . For researchers, understanding PRKD1's function is essential as it represents a potential therapeutic target and diagnostic marker, particularly in breast cancer where its epigenetic silencing correlates with increased tumor invasiveness and metastatic potential .

What are the recommended applications for PRKD1 antibodies in experimental design?

PRKD1 antibodies are versatile tools applicable across multiple experimental platforms. Based on validation data, these antibodies are particularly effective in:

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Recommended dilution ranges from 1:50-1:500, with positive detection demonstrated in HeLa and LNCaP cell lines .

• Flow Cytometry (Intracellular): Optimal concentration is 0.25 μg per 10^6 cells in a 100 μl suspension, with validated detection in HeLa cells .

• ELISA applications: While specific dilutions may vary by experimental design, PRKD1 antibodies have shown reliability in this format .

How should researchers validate PRKD1 antibody specificity before experimental use?

Validation of PRKD1 antibody specificity is a critical preliminary step that should incorporate multiple complementary approaches:

• Western Blot Analysis: Confirm detection of the expected 102 kDa band corresponding to PRKD1 protein. Comparison with positive and negative control cell lines is essential.

• Knockout/Knockdown Controls: Utilize PRKD1 knockout or knockdown cell models as negative controls to verify antibody specificity. Research has demonstrated the effectiveness of this approach in PRKD1 studies, where knockdown models showed significant phenotypic changes validating antibody specificity .

• Cross-Reactivity Assessment: For biotin-conjugated antibodies specifically, researchers should perform additional controls to ensure the biotin conjugation doesn't create non-specific binding or cross-reactivity with biotin-binding proteins naturally present in tissues.

• Peptide Competition Assay: Consider performing a peptide competition assay using the immunogenic peptide (such as the 21-amino acid peptide KSPESFIGREKRSNSQSYIG from the N-terminal of human PRKD1 ) to confirm binding specificity.

What are the optimal storage and handling procedures for maintaining PRKD1 antibody activity?

Long-term performance of PRKD1 antibodies depends on proper storage and handling practices:

For biotin-conjugated PRKD1 antibodies, additional precautions may be necessary to preserve both antibody activity and biotin functionality. Researchers should avoid exposure to strong light and oxidizing agents that could compromise the biotin moiety .

How does epigenetic regulation of PRKD1 impact antibody-based detection methods?

Epigenetic silencing of PRKD1 through promoter methylation presents unique challenges for antibody-based detection:

• Variable Expression Levels: Research has demonstrated that PRKD1 gene expression is dramatically decreased in advanced breast tumors due to promoter methylation . This variable expression across different tissue types and disease states requires careful optimization of antibody concentrations.

• Detection Sensitivity Considerations: In samples with epigenetic silencing of PRKD1, researchers may need to employ signal amplification methods to detect low-abundance protein. Biotin-conjugated antibodies offer an advantage in this context due to their compatibility with streptavidin-based amplification systems.

• Experimental Controls: When studying PRKD1 in cancer tissues, researchers should consider including matched normal-tumor pairs and methylation status analysis alongside antibody detection. Studies have shown that using DNA methyltransferase inhibitors like decitabine can restore PRKD1 expression, providing useful positive controls for antibody validation in epigenetically silenced contexts .

• Interpretation Challenges: Low signal in immunodetection experiments could indicate either technical failure or biological downregulation of PRKD1. Complementary assessment of PRKD1 promoter methylation status using methylation-specific PCR can help distinguish between these possibilities .

What are the critical considerations when using PRKD1 antibodies for studying cancer progression?

PRKD1's role in cancer progression requires specific experimental approaches:

• Cell Line Selection: Research has shown significant differences in PRKD1 expression between invasive (e.g., MDA-MB-231) and non-invasive (e.g., MCF-7) breast cancer cell lines . Careful selection of appropriate cell models is essential for meaningful results.

• Functional Assessment: Beyond simple detection, PRKD1 antibodies can be utilized in functional studies to correlate protein expression with invasive phenotypes. Transwell invasion assays following manipulation of PRKD1 expression have demonstrated the protein's role in suppressing invasion .

• In Vivo Considerations: Animal models have validated the relationship between PRKD1 expression and metastatic potential. PRKD1 antibodies can be employed in tissue analyses from such models, particularly when assessing the effects of interventions like decitabine treatment that restore PRKD1 expression .

• Signaling Pathway Integration: PRKD1 intersects with multiple signaling pathways relevant to cancer progression. When designing experiments, researchers should consider co-staining for pathway components such as pCreb1, Yap1, and c-Myc, which have been shown to be affected by PRKD1 expression levels .

What are common issues encountered in immunofluorescence applications with PRKD1 antibodies and their solutions?

Immunofluorescence protocols require careful optimization when using PRKD1 antibodies:

For biotin-conjugated PRKD1 antibodies specifically, researchers should be aware of potential endogenous biotin in certain tissues, which may necessitate additional blocking steps with avidin/biotin blocking kits before antibody application.

How can researchers optimize flow cytometry protocols when using PRKD1 antibodies?

Flow cytometry with PRKD1 antibodies requires attention to several key factors:

• Cell Fixation and Permeabilization: As PRKD1 is primarily an intracellular target, thorough permeabilization is essential. A standardized protocol using 0.25 μg of antibody per 10^6 cells in a 100 μl suspension has been validated for intracellular flow cytometry in HeLa cells .

• Antibody Titration: Despite recommended starting concentrations, researchers should perform antibody titration experiments to determine the optimal signal-to-noise ratio for their specific cell types and equipment.

• Compensation Controls: For biotin-conjugated antibodies paired with fluorochrome-conjugated streptavidin, proper compensation controls are critical, especially in multi-parameter experiments.

• Analysis Considerations: When analyzing PRKD1 expression levels, researchers should establish clear positive and negative population boundaries based on control samples, particularly when examining heterogeneous clinical specimens.

What methodological approaches can resolve discrepancies between PRKD1 protein and mRNA expression levels?

Research has revealed complex regulatory mechanisms affecting PRKD1 expression:

• Post-transcriptional Regulation: Studies have identified regulatory elements like the miR-17 motif that can affect mRNA stability and translation efficiency of genes like PKD1 . Similar mechanisms might affect PRKD1, creating discrepancies between transcript and protein levels.

• Integrated Analysis Approach: Researchers should combine RT-PCR (using primers such as 5′-TTCTCCCACCTCAGGTCATC-3′ and 5′-TGCCAGAGCACATAACGAAG-3′ for human PRKD1 ) with protein detection via immunoblotting or immunohistochemistry.

• Epigenetic Assessment: Include methylation-specific PCR of the PRKD1 promoter (using primers like 5′-AGAGGGTTAGTCGGGTAGC-3′ and 5′-ACGTCCGCGAAATAACTTA-3′ for methylated promoter detection ) to assess epigenetic silencing as a cause of expression discrepancies.

• Translation Efficiency Analysis: When discrepancies persist, consider analyzing polysome profiles to assess translation efficiency of PRKD1 mRNA, as post-transcriptional regulatory mechanisms may affect protein synthesis independent of mRNA levels.

How can PRKD1 antibodies contribute to understanding epigenetic regulation in cancer progression?

PRKD1 antibodies serve as valuable tools for exploring epigenetic mechanisms in cancer:

• Therapeutic Response Monitoring: Research has demonstrated that treatment with DNA methyltransferase inhibitors like decitabine can reverse PRKD1 promoter methylation and restore protein expression . PRKD1 antibodies enable researchers to monitor this reexpression at the protein level.

• Biomarker Development: Studies suggest that the epigenetic regulation status of the PRKD1 promoter could serve as an early diagnostic marker for breast tumor invasiveness . Antibody-based detection methods can translate these findings into clinical applications.

• Mechanistic Studies: By combining PRKD1 antibody detection with analysis of downstream signaling pathways, researchers can elucidate the molecular mechanisms through which PRKD1 suppresses invasive phenotypes in cancer cells.

• In Vivo Validation: Animal studies have confirmed that restoration of PRKD1 expression can block tumor spread and metastasis in a PKD1-dependent manner . Immunohistochemical analysis using PRKD1 antibodies provides crucial validation of these effects in tissue contexts.

What strategies can enhance specificity when using PRKD1 antibodies in complex tissue samples?

Working with heterogeneous tissue samples presents unique challenges:

• Multiplex Immunostaining: Combine PRKD1 antibodies with markers for specific cell types to distinguish expression patterns within complex tissues. This approach is particularly valuable in tumor microenvironments where expression may vary between malignant cells and stromal components.

• Signal Amplification Methods: For biotin-conjugated antibodies, employ tyramide signal amplification or multiple-layer detection systems to enhance sensitivity while maintaining specificity in tissues with low PRKD1 expression.

• Background Reduction Techniques: Implement tissue-specific blocking methods, such as FcR blocking for lymphoid tissues or endogenous biotin blocking when using biotin-conjugated antibodies.

• Validation with Alternative Methods: Confirm immunohistochemistry findings with orthogonal techniques such as in situ MSP-PCR, which has been successfully used to analyze PRKD1 promoter methylation in breast cancer tissues .

How does PRKD1 investigation intersect with other kinase research pathways?

PRKD1 functions within a complex network of signaling pathways:

• Differential Analysis: When studying PRKD1, researchers should consider its interactions with related family members (PKD2, PKD3) and distinguish these from the unrelated PKD1 (Polycystin-1) protein associated with polycystic kidney disease .

• Pathway Integration: Research has shown that PRKD1 expression affects levels of signaling molecules including Yap1, pCreb1, and c-Myc . Antibody-based multiplexing approaches can reveal these interconnections.

• Therapeutic Implications: As kinase pathways represent important therapeutic targets, PRKD1 antibodies enable researchers to monitor pathway modulation in response to experimental therapeutics, particularly those targeting epigenetic mechanisms.

• Translational Applications: The documented role of PRKD1 in suppressing cancer cell invasion suggests potential for translational applications where antibody-based detection could inform clinical decision-making regarding invasive potential or metastatic risk.

What emerging technologies might enhance PRKD1 antibody applications in research?

The landscape of PRKD1 research continues to evolve with emerging technologies:

• Single-Cell Analysis: Adaptation of PRKD1 antibodies for single-cell proteomic techniques will enable more nuanced understanding of expression heterogeneity within tumors and other complex tissues.

• Proximity Ligation Assays: These techniques can reveal protein-protein interactions involving PRKD1, providing insights into its functional partners and regulatory mechanisms.

• Live-Cell Imaging Applications: Development of non-disruptive labeling techniques using PRKD1 antibody fragments could enable real-time monitoring of protein dynamics.

• Therapeutic Development: Beyond research applications, PRKD1 antibodies may contribute to therapeutic development through target validation studies and potential applications in antibody-drug conjugates targeting cells with aberrant PRKD1 expression.