PKN1 Antibody

What applications are most reliable for PKN1 antibody detection?

PKN1 antibodies have been validated for several experimental applications with varying degrees of reliability. Western blot analysis has consistently shown high specificity for detecting the 120 kDa PKN1 protein in human, mouse, and rat samples . Immunoprecipitation has also proven effective, particularly when using monoclonal antibodies like EPR18808 at 1/40 dilution with Jurkat cell lysates . For tissue-specific detection, immunohistochemistry provides reliable results, though optimization of antigen retrieval methods is essential. ELISA applications require careful validation but have been successful with specific antibody preparations .

When selecting applications, consider that:

• Western blot is optimal for protein size verification

• Immunoprecipitation is valuable for studying protein-protein interactions

• Immunohistochemistry provides spatial context in tissues

How should PKN1 antibodies be validated for experimental specificity?

Comprehensive validation of PKN1 antibody specificity requires multiple approaches:

• Knockout validation: The gold standard involves testing antibodies on PKN1 knockout samples. As demonstrated with ab195264, when PKN1 knockout samples were subjected to SDS-PAGE alongside wild-type samples, the antibody specifically detected PKN1 only in wild-type samples while showing no reactivity in knockout samples .

• Multiple antibody comparison: Using antibodies targeting different epitopes of PKN1 can confirm specificity. Available antibodies target various regions including:

  • EPR18808: N-terminal region

  • Clone 540421: Ile215-Thr388 region

  • Clone 1B10: Peptide sequence containing LDMEPQGCLVAEVTFRNPVI

• Cellular context validation: Testing across multiple cell lines is essential. PKN1 antibodies have been verified in:

  • HeLa (human cervical epithelial carcinoma cells)

  • L1.2 (mouse pro-B cells)

  • Jurkat (human T cell leukemia cells)

What are the critical differences between monoclonal and polyclonal PKN1 antibodies for research applications?

Research has demonstrated that monoclonal antibodies provide more consistent results for co-immunoprecipitation studies examining PKN1's interaction with proteins like Frizzled 7 receptor , while polyclonal antibodies may offer advantages for cross-species detection and when protein conformation is altered.

How can PKN1 phosphorylation status be effectively monitored using antibody-based techniques?

Monitoring PKN1 phosphorylation requires specialized approaches as several phosphorylation sites are functionally significant:

• Site-specific phospho-antibodies: The S374 phosphorylation site has been identified as functionally relevant for axonal outgrowth and AKT interaction . Researchers should use phospho-specific antibodies targeting PKN1-pS374 for direct detection.

• Temporal assessment: PKN1 phosphorylation shows developmental regulation. Research demonstrates that PKN1-pS374 exhibits a steep decrease during cerebellar development , necessitating time-course experiments for accurate interpretation.

• Combined phospho-protein analysis: Since PKN1 functions within phosphorylation cascades, parallel monitoring of downstream targets provides functional context:

  • Monitor AKT phosphorylation status

  • Assess GSK3β phosphorylation

  • Examine p70S6Kinase activation levels

• Experimental validation: Confirm phospho-antibody specificity using:

  • Phosphatase treatment controls

  • PKN1 knockout tissues

  • PKN1 T778A kinase-negative mutant cells for comparison

The functional relationship between PKN1 phosphorylation and downstream signaling can be effectively visualized using immunoblotting time course studies, particularly during hypoxia-reperfusion studies where temporal dynamics are critical .

What approaches are recommended for investigating PKN1's role in Wnt/β-catenin signaling?

Investigation of PKN1's inhibitory role in Wnt/β-catenin signaling requires specialized experimental approaches:

• PKN1 knockdown studies: siRNA-mediated PKN1 reduction significantly enhances activation of β-catenin-activated reporter and increases apoptosis in melanoma cell lines . Effective knockdown validation requires:

  • Confirmation of PKN1 reduction by Western blot

  • Assessment of multiple siRNA sequences to rule out off-target effects

  • Quantitative PCR validation of knockdown efficiency

• Protein complex analysis: PKN1 forms a complex with Wnt3A receptor Frizzled 7. This interaction can be studied through:

  • Affinity purification followed by mass spectrometry

  • Co-immunoprecipitation using PKN1 antibodies

  • Verification of interactions using reciprocal pull-downs

• Functional readouts:

  • β-catenin reporter assays

  • Apoptosis measurements in melanoma cell lines

  • Phosphorylation status of β-catenin

Research has shown that at least two independent affinity purifications should be performed, and only proteins identified by two independent peptides in one preparation should be considered for further analysis . This stringent approach helps eliminate false positives in protein interaction studies.

How can PKN1 antibodies be applied to study the autoimmune-like phenotypes in animal models?

PKN1 has demonstrated significant roles in autoimmune-like phenotypes, requiring specific antibody-based approaches:

• Comparative phenotype analysis: PKN1 knockout (KO) mice develop an autoimmune-like disease with age, showing spontaneous germinal center (GC) formation, autoantibody production, and glomerulonephritis . In contrast, PKN1[T778A] kinase-negative knock-in mice show different phenotypes:

  • Splenomegaly and leukocyte trafficking impairment

  • No obvious GC formation

  • No autoimmune-like symptoms such as glomerulonephritis

• Flow cytometric analysis protocols: For spleen studies, analyze:

  • B220+ (B cell marker)

  • CD3+ (T cell marker)

  • Gr1+ and CD193+ (granulocyte markers)

  • CD19+CD95+GL7+ (GC B cell fraction)

• Histological verification:

  • H&E staining of spleen sections

  • Examination of red pulp expansion

  • Assessment of increased cellularity

• Molecular analysis:

  • Southern blot analysis for enhanced monoclonal rearrangements of IgH gene J region

  • TCR β gene C region to detect potential lymphomagenesis

Research findings indicate that PKN1 may have both kinase-dependent and kinase-independent functions in vivo, with distinct roles in autoimmune processes that should be discriminated using both knockout and kinase-negative models .

How should researchers address discrepancies between antibody-based PKN1 detection and gene expression data?

When faced with discrepancies between PKN1 protein levels (antibody detection) and gene expression data, consider these methodological approaches:

• Verify antibody specificity: Ensure the PKN1 antibody detects the correct protein by:

  • Testing on PKN1 knockout samples

  • Using multiple antibodies targeting different epitopes

  • Confirming the expected molecular weight (~120 kDa)

• Consider post-transcriptional regulation: Research has shown that PKN1 undergoes significant post-transcriptional regulation, including:

  • Protein stability changes through ubiquitination

  • Proteolytic activation by caspase-3 during apoptosis

  • Cellular compartmentalization that may affect extraction efficiency

• Validate expression analysis methods:

  • For qPCR, ensure primers span exon-exon junctions

  • For RNA-seq, check for alternative splicing events that might affect antibody binding sites

  • Use multiple reference genes for normalization

• Technical considerations:

  • Sample preparation methods may differentially affect protein versus RNA integrity

  • Antibody detection may be influenced by protein modifications or complex formation

  • Confirm antibody lot consistency if experiments were performed across different timepoints

A systematic approach addressing these factors can help reconcile discrepancies and provide more accurate insights into PKN1 biology.

What controls are essential when using PKN1 antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (co-IP) experiments investigating PKN1 protein interactions require rigorous controls:

• Essential negative controls:

  • IgG isotype control: Use matching host species IgG for non-specific binding assessment

  • PKN1 knockout or knockdown samples: Demonstrates specificity of interactions

  • Reciprocal IP: Confirm interaction by pulling down with antibodies against the suspected interacting partner

• Positive controls:

  • Input sample: Include 5-10% of pre-IP lysate

  • Known PKN1 interactors: Include established partners like Frizzled 7 receptor, which has been validated by multiple methods

  • Recombinant PKN1 protein: For antibody validation

• Technical validation:

  • Multiple antibody approach: Use antibodies targeting different PKN1 epitopes

  • Stringency testing: Perform washes with increasing salt concentrations to determine interaction strength

  • Crosslinking considerations: Determine if transient interactions require crosslinking

• Experimental design specifics:

  • Lysate preparation: 1mg of whole cell lysate (as used with Jurkat cells)

  • Antibody concentration: 1/40 dilution demonstrated effective for EPR18808

  • Verification methods: Follow IP with Western blot or mass spectrometry

For investigating novel PKN1 interactions, validation through at least two independent affinity purifications is recommended, with identification by two independent peptides in at least one preparation .

How can researchers effectively study PKN1 in neurological disease models using antibody-based approaches?

PKN1 has demonstrated important roles in neurological conditions, particularly in hypoxic-ischemic encephalopathy models:

• Cerebellar granule cell (Cgc) culture system:

  • PKN1 exerts neurodegenerative effects via inhibition of AKT/GSK3β signaling

  • Pkn1-/- Cgc shows higher AKT phosphorylation upon hypoxia-ischemia (HI)

  • Enhanced phosphorylation of downstream targets including p70S6Kinase and GSK3β

  • Reduced cleavage of caspase-3

• Antibody-based detection protocol:

  • Monitor PKN1-pS374, which shows developmental regulation

  • Assess AKT phosphorylation status during early reperfusion periods

  • Examine caspase-3 activation during longer reperfusion periods (3h OGD, 24h Rep)

• Axonal outgrowth assessment:

  • PKN1-/- cerebellar granule cells show enhanced axonal outgrowth on laminin

  • Testing on chondroitin sulfate proteoglycans (CSPGs) substrates reveals growth-inhibitory glial scar effects

  • S374 phosphorylation site is functionally relevant for axonal outgrowth and AKT interaction

• Methodology specifics:

  • Western blotting for phosphorylation cascade analysis

  • Immunocytochemistry for assessing axonal outgrowth

  • Live/dead cell staining for viability assessment post-HI

Research indicates that post-ischemic AKT hyperactivation upon Pkn1 knockout is particularly relevant during early reperfusion, while the protective effect on caspase-3 activation persists during longer reperfusion periods .

What are the recommended approaches for using PKN1 antibodies across different animal models?

When studying PKN1 across different species, researchers should consider these validated approaches:

• Species-specific considerations:

  • Human and mouse PKN1 antibodies: Many antibodies show cross-reactivity between human and mouse PKN1, such as clone 540421 which recognizes Ile215-Thr388 of human PKN1

  • Non-human primate models: PKN1 antibodies have been validated in macaque studies examining Chlamydia trachomatis infection

  • Rodent models: Several antibodies demonstrate reactivity with rat PKN1

• Validation methods across species:

  • Sequence homology analysis of the targeted epitope

  • Testing on tissues from multiple species

  • Positive and negative controls for each species

• Application-specific protocols:

  • For Western blot: Human (HeLa), mouse (L1.2) cell lines have been validated

  • For ELISA: Validation in both human patient samples and macaque models demonstrated for PKN1

  • For immunohistochemistry: Tissue-specific optimization required for each species

• Experimental design considerations:

  • Age-dependent changes: PKN1[T778A] mice show age-dependent phenotypes, with spleen size increasing significantly in aged mice

  • Developmental regulation: PKN1-pS374 shows steep decrease during cerebellar development

  • Time-course studies: Especially important for autoimmune phenotypes that develop with age (>30 weeks)

Research has demonstrated that antibodies recognizing Ile215-Thr388 of human PKN1 can be effective across species, while epitope-specific antibodies may require sequence verification before cross-species application .

How can PKN1's role in splenomegaly and immunological disorders be investigated using antibody-based techniques?

Investigation of PKN1's role in splenomegaly and immunological disorders requires specialized approaches:

• Flow cytometric analysis protocol:

  • Assess spleen leukocyte populations using markers:

    • B220+ (B cells)

    • CD3+ (T cells)

    • Gr1+ and CD193+ (granulocytes)

    • CD19+CD95+GL7+ (germinal center B cells)

  • Compare with peripheral blood leukocyte counts

• Histological assessment:

  • H&E staining for red pulp expansion and increased cellularity

  • Section analysis for extramedullary hematopoiesis

  • Examination for spontaneous germinal center formation

• Molecular characterization:

  • Southern blot analysis for monoclonal rearrangements in IgH gene J region

  • TCR β gene C region evaluation for lymphomagenesis

  • Antibody titer measurements (anti-dsDNA antibodies)

• PKN1 model comparison:

  • Contrasting phenotypes between PKN1 knockout versus PKN1[T778A] kinase-negative models:

    • PKN1 knockout: Spontaneous GC formation, autoimmune-like disease, autoantibody production

    • PKN1[T778A]: Splenomegaly without GC formation or autoimmune phenotypes

Research findings indicate that complete PKN1 knockout versus kinase-inactivation produces different immunological outcomes, suggesting kinase-independent functions of PKN1 in immune regulation. PKN1[T778A] mice showed enlarged spleens (0.33g/1.13% of body weight compared to 0.14g/0.48% in wild type) and higher incidence of enlarged lymph nodes, but without the autoimmune features seen in complete knockout models .

How can PKN1 antibodies contribute to cancer research, particularly in melanoma studies?

PKN1 antibodies offer valuable tools for investigating its role in cancer, particularly in melanoma where it inhibits Wnt/β-catenin signaling:

• Signaling pathway analysis:

  • PKN1 represses Wnt/β-catenin signaling in melanoma cells

  • siRNA-mediated PKN1 reduction enhances β-catenin-activated reporter activity

  • PKN1 knockdown increases apoptosis in melanoma cell lines

• Protein complex characterization:

  • Affinity purification followed by mass spectrometry reveals PKN1 in a complex with WNT3A receptor Frizzled 7

  • Co-purification studies identify novel protein interactions

  • Experimental protocol: Two independent affinity purifications with identification by two independent peptides in at least one preparation

• Phosphoproteomics integration:

  • Phosphoproteomic screens can identify PKN1-mediated phosphorylation events

  • RNA interference screens can be integrated to identify functional significance

  • Creating protein-protein interaction networks using STRING database, BioGRID, and Human Protein Reference Database

• Methodological considerations:

  • Western blotting for PKN1 expression levels in cancer cells

  • Immunohistochemistry for tissue distribution patterns

  • β-catenin reporter assays for functional assessment of PKN1 manipulation

PKN1 antibodies enable researchers to investigate its overexpression in various carcinomas, with particular relevance in ovarian serous carcinoma where PRK1/PKN1 has been characterized in detail .

What considerations are important when using PKN1 antibodies for vaccine candidate research?

PKN1 has emerged as a potential vaccine candidate in certain infectious disease contexts, particularly in Chlamydia trachomatis research:

• Seroprevalence assessment:

  • ELISA protocols for detecting antibodies against PKN1 in:

    • Human patient sera

    • Experimental animal models (Macaca nemestrina)

    • Cut-off value determination: Mean ± (3X S.D.) of healthy samples

• Recombinant protein production for immunological studies:

  • Cloning of Pkn1 (1.7 kb) from C. trachomatis genomic DNA

  • Expression and purification using Ni+-NTA chromatography

  • Elution at 300 mM imidazole

  • Verification by Coomassie staining and Western blot with anti-his antibodies

• Epitope identification:

  • Bioinformatic analysis using BCePred software

  • Top scoring epitopes for PKN1 present towards C-terminal

  • Careful analysis of solvent-accessible regions (typically N- and C-terminal)

• Comparative seroreactivity assessment:

  • PKN1 shows 67% seroreactivity in C. trachomatis-infected macaques

  • Significant prevalence of anti-PKN1 antibodies in infected humans

  • Statistical analysis using Spearman's rank method to find correlations between antichlamydial antigens