PKN2 Recombinant Monoclonal Antibody

What is PKN2 and what are its primary biological functions?

PKN2 (also known as PRK2, PRKCL2, or Protein kinase C-like 2) is a PKC-related serine/threonine-protein kinase that functions as a Rho/Rac effector protein participating in specific signal transduction responses. PKN2 plays critical roles in multiple cellular processes including regulation of cell cycle progression, actin cytoskeleton assembly, cell migration, cell adhesion, and tumor cell invasion. It also participates in transcription activation signaling processes that are essential for normal cellular function. As a direct RhoA target, PKN2 is required for the regulation of primordial junctions maturation into apical junction formation in bronchial epithelial cells, highlighting its importance in maintaining cellular architecture and communication .

What cell types and tissues show significant PKN2 expression?

PKN2 expression has been confirmed in multiple cell lines and tissue types. The protein is expressed in human liver hepatocellular carcinoma cell lines (HepG2), human cervical cancer cells (HeLa), human embryonic kidney cells (HEK-293/293T), human colorectal cancer cells (COLO 320), human ovarian cancer cells (SKOV-3, A2780), and human chronic myelogenous leukemia cells (K-562). In terms of tissues, PKN2 expression has been detected in human stomach tissue and human colon cancer tissue through immunohistochemistry (IHC) analysis . This widespread expression pattern indicates PKN2's fundamental role in cellular physiology across diverse tissue types and suggests its potential involvement in various disease states, particularly in cancer progression.

What are the recommended applications for PKN2 Recombinant Monoclonal Antibodies?

PKN2 Recombinant Monoclonal Antibodies have been validated for multiple research applications with specific recommended dilutions for optimal results:

For IHC applications, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may also be effective . When optimizing experimental conditions, it's recommended to perform antibody titration as results may vary depending on specific sample types and experimental systems.

How do recombinant monoclonal antibodies against PKN2 differ from polyclonal alternatives?

Recombinant monoclonal antibodies against PKN2, such as the EPR5490 clone, offer several distinct advantages over polyclonal alternatives. Recombinant monoclonal antibodies are produced through recombinant DNA technology, ensuring consistent epitope recognition across different production lots. This results in higher reproducibility and reliability for long-term research projects. In contrast, polyclonal antibodies recognize multiple epitopes but may show batch-to-batch variation that can complicate result interpretation .

What are the optimal storage and handling conditions for PKN2 recombinant monoclonal antibodies?

For maximum stability and performance of PKN2 recombinant monoclonal antibodies, proper storage and handling protocols are essential. These antibodies are typically supplied in PBS buffer with additives such as 0.05% BSA, 50% glycerol, and 0.02% sodium azide at pH 7.3 to maintain stability .

For long-term storage, antibodies should be kept at -20°C, avoiding repeated freeze-thaw cycles which can lead to protein denaturation and loss of binding activity. For short-term use (up to one month), storage at 4°C is acceptable. When working with these antibodies, it's advisable to aliquot the stock solution upon first thawing to minimize freeze-thaw cycles. Additionally, all handling should be done using sterile techniques to prevent microbial contamination. Prior to each use, gently mix the antibody solution by inversion rather than vortexing to prevent protein aggregation that could compromise antibody function and lead to non-specific binding in experimental applications .

What controls should be included when using PKN2 antibodies in experimental procedures?

Including appropriate controls is crucial for ensuring the validity and interpretability of results when using PKN2 antibodies. For Western blotting applications, the following controls should be implemented:

• Positive controls: Cell lysates with confirmed PKN2 expression (e.g., HeLa, HEK-293T, COLO 320) should be included to verify antibody functionality .

• Negative controls: PKN2 knockout cell lysates, such as those from PKN2 knockout HeLa cell line (ab264691) or PKN2 knockout HCT116 cell line (ab266894), provide essential verification of antibody specificity .

• Loading controls: Use of housekeeping proteins like GAPDH (with antibodies such as ab8245) ensures equal protein loading across samples and allows for proper quantification .

For immunohistochemistry or immunofluorescence applications, additional controls should include:

• Secondary antibody-only controls to assess background staining

• Isotype controls to evaluate non-specific binding

• Peptide competition assays where feasible to confirm binding specificity

These controls collectively help distinguish true positive signals from artifacts or non-specific binding, enabling confident interpretation of experimental results .

How can PKN2 antibodies be effectively used to study its role in cancer progression and metastasis?

PKN2 has emerged as a significant player in cancer progression, particularly through its involvement in cell migration, adhesion, and tumor cell invasion. To investigate these roles, researchers can employ multiple complementary approaches using PKN2 antibodies:

For analyzing PKN2 expression levels across different cancer stages, immunohistochemistry using PKN2 antibodies (dilution 1:200-1:1000) on tissue microarrays can provide valuable insights into correlation with clinical outcomes. This approach has been successfully applied to human colon cancer tissue samples . Western blot analysis (1:500-1:3000 dilution) comparing PKN2 expression in matched normal and tumor tissue samples or across cell lines with varying metastatic potential can reveal expression patterns associated with disease progression .

To study PKN2's functional role in cancer metastasis, researchers can combine PKN2 antibodies with other methodologies. Immunofluorescence microscopy using PKN2 antibodies (1:10-1:100) can visualize its subcellular localization during cell migration and invasion processes. Co-immunoprecipitation studies using PKN2 antibodies (0.5-4.0 μg for IP) can identify binding partners specific to metastatic contexts, potentially revealing signaling nodes that could be therapeutically targeted . These approaches, when complemented with gene knockdown or knockout models, provide a comprehensive understanding of PKN2's mechanistic contributions to cancer progression.

What approaches can be used to investigate PKN2's interaction with the RhoA signaling pathway?

PKN2 functions as a direct effector of RhoA signaling, making the investigation of this interaction crucial for understanding cytoskeletal dynamics and cell junction formation. To elucidate these interactions, several sophisticated approaches can be employed:

Proximity ligation assays (PLA) using PKN2 monoclonal antibodies in conjunction with RhoA antibodies can visualize direct protein-protein interactions in situ, providing spatial and temporal information about when and where these proteins interact within the cell. For co-immunoprecipitation studies, PKN2 antibodies can be used to pull down protein complexes (0.5-4.0 μg per 1-3 mg protein lysate), followed by Western blotting for RhoA and other potential binding partners .

To explore the functional consequences of these interactions, researchers can combine PKN2 immunofluorescence (1:10-1:100 dilution) with RhoA activity sensors in live-cell imaging experiments. This approach allows simultaneous visualization of PKN2 localization and RhoA activation states during processes like junction formation in epithelial cells. Additionally, phosphoproteomic analysis following RhoA activation, coupled with PKN2 immunoprecipitation, can identify PKN2 substrates within the RhoA signaling cascade, providing a more comprehensive understanding of this signaling network .

How can PKN2 antibodies be applied in studying cell cycle regulation and cytokinesis?

PKN2 plays a critical role in G2/M phase progression and cytokinesis, making it an important target for cell cycle research. To investigate these functions, researchers can implement several sophisticated experimental approaches:

For studying PKN2's temporal dynamics during cell cycle progression, synchronization of cells at different cell cycle stages followed by immunofluorescence microscopy (IF/ICC at 1:10-1:100 dilution) can reveal changes in PKN2 localization. This approach is particularly valuable during mitosis and cytokinesis, where PKN2 demonstrates distinct localization patterns. Flow cytometry with dual staining for PKN2 (0.40 μg per 10^6 cells) and DNA content can quantitatively assess PKN2 levels across different cell cycle phases in large cell populations .

To understand PKN2's functional role in cytokinesis, live-cell imaging combined with PKN2 immunofluorescence can visualize its interaction with the cytokinetic machinery. Additionally, immunoprecipitation of PKN2 (0.5-4.0 μg for IP) from cells arrested at the cytokinesis stage, followed by mass spectrometry analysis, can identify cytokinesis-specific interaction partners. Researchers can also employ phospho-specific antibodies against known PKN2 substrates to track the activation of downstream signaling during cell cycle progression. These approaches collectively provide mechanistic insights into how PKN2 contributes to proper cell division and genomic stability .

How can non-specific binding be minimized when using PKN2 antibodies in immunohistochemistry?

Non-specific binding is a common challenge in immunohistochemistry that can compromise result interpretation. For PKN2 antibodies, several optimization strategies can minimize this issue:

Optimizing blocking conditions is crucial—using 5-10% normal serum from the same species as the secondary antibody for 1-2 hours at room temperature can effectively block non-specific binding sites. For tissues with high endogenous biotin, implementing an avidin-biotin blocking step can significantly reduce background. Additionally, titrating the PKN2 antibody concentration is essential—starting with the recommended dilution range (1:200-1:1000) and adjusting based on signal-to-noise ratio improves specificity .

Proper antigen retrieval is particularly important for PKN2 detection. While TE buffer at pH 9.0 is recommended, comparing results with citrate buffer at pH 6.0 may be necessary to determine optimal conditions for specific tissue types. If background persists, increasing washing steps (3-5 washes of 5 minutes each) with TBS-T (0.1% Tween-20) and adding 0.1-0.3% Triton X-100 to the antibody diluent can improve results. Finally, including PKN2 knockout tissue sections as negative controls provides definitive confirmation of staining specificity .

What are the common pitfalls when detecting PKN2 by Western blotting and how can they be addressed?

Western blotting for PKN2 can present several technical challenges due to its high molecular weight (predicted 112 kDa, sometimes observed at 125 kDa). To achieve optimal results, researchers should address these common pitfalls:

For complete protein transfer of high-molecular-weight PKN2, extended transfer times or lower voltage settings are recommended. Using gradient gels (4-12% or 4-15%) rather than fixed-percentage gels improves separation of PKN2 from similarly sized proteins. When loading samples, balancing protein concentration is critical—too much protein can cause smearing while too little leads to weak signal. The recommended loading amount is 15-30 μg of total protein per lane, with antibody dilutions of 1:500-1:3000 .

Several validation studies report observing PKN2 at band sizes slightly different from the predicted 112 kDa (sometimes at 125 kDa), which may reflect post-translational modifications or isoform expression. To confirm band specificity, researchers should compare results with PKN2 knockout cell lysates (such as from PKN2 knockout HeLa cells) as negative controls. Additionally, optimization of membrane blocking (5% non-fat milk or BSA in TBS-T for 1 hour) and incubation temperatures (4°C overnight versus room temperature for 1-2 hours) can significantly improve signal-to-noise ratio for PKN2 detection .

What approaches can resolve inconsistent results when using PKN2 antibodies across different experimental systems?

When researchers encounter variability in PKN2 antibody performance across different experimental systems, several methodical approaches can help identify and resolve these inconsistencies:

First, performing antibody validation using multiple techniques is essential. If an antibody works well in Western blot but poorly in IHC, this may indicate epitope masking in fixed tissues. Comparing results from different PKN2 antibody clones recognizing distinct epitopes can provide complementary data and confirm findings. Additionally, cross-validation with orthogonal methods—such as correlating protein detection with mRNA expression data or using genetic approaches (siRNA, CRISPR)—strengthens confidence in antibody specificity .

Cell type-specific factors can significantly influence results. PKN2 undergoes post-translational modifications that may vary across cell types, potentially affecting antibody recognition. Researchers should optimize fixation conditions for each cell type or tissue, as overfixation can mask epitopes while underfixation may compromise tissue morphology. When inconsistencies persist, implementing a systematic optimization approach—testing different antibody lots, varied incubation times, alternative buffers, and detection systems—can identify optimal conditions for each experimental system. Documenting these optimization steps is crucial for result reproducibility and interpretation .

How might PKN2 antibodies contribute to understanding its role in therapeutic resistance mechanisms?

PKN2's involvement in cell survival pathways, particularly through its inhibition of Akt pro-survival-induced kinase activity, positions it as a potential mediator of therapeutic resistance in cancer. PKN2 antibodies can be instrumental in elucidating these mechanisms through several sophisticated approaches:

Researchers can employ PKN2 antibodies in immunohistochemistry (1:200-1:1000) to analyze PKN2 expression patterns in matched pre- and post-treatment patient samples, potentially identifying expression changes associated with treatment resistance. Combining PKN2 antibodies with phospho-specific antibodies against key signaling nodes in resistance pathways (such as phospho-Akt, phospho-ERK) in multiplexed immunofluorescence can reveal altered signaling networks in resistant cells .

For mechanistic studies, co-immunoprecipitation using PKN2 antibodies (0.5-4.0 μg for IP) followed by mass spectrometry can identify novel interaction partners that emerge during resistance development. In cell line models of acquired resistance, PKN2 immunoprecipitation coupled with phosphoproteomic analysis can map alterations in PKN2-dependent phosphorylation cascades. These approaches, when combined with pharmacological inhibition or genetic modulation of PKN2, may uncover targetable vulnerabilities in resistant cancer cells, potentially informing the development of more effective combination therapies .

What emerging techniques could enhance the utility of PKN2 antibodies in studying its domain-specific functions?

Emerging technological approaches are expanding the potential applications of PKN2 antibodies for investigating domain-specific functions of this multifunctional protein. These innovative techniques offer new insights into PKN2 biology:

Super-resolution microscopy techniques (STED, STORM, PALM) combined with domain-specific PKN2 antibodies can provide unprecedented spatial resolution of PKN2 localization within subcellular compartments, revealing previously undetectable interaction domains. For studying dynamic PKN2 functions, CRISPR-based tagging of endogenous PKN2 with split fluorescent proteins, complemented by antibody validation, enables real-time visualization of domain-specific interactions in living cells .

Advanced proximity labeling approaches such as BioID or APEX2 fused to specific PKN2 domains, followed by detection with PKN2 antibodies, can map domain-specific protein interaction networks with high spatial and temporal resolution. Additionally, hydrogen-deuterium exchange mass spectrometry combined with PKN2 antibody-based purification can identify conformational changes in PKN2 following activation by different upstream signals. These techniques, collectively, provide more nuanced understanding of how PKN2's multiple domains coordinate its diverse cellular functions, potentially revealing novel regulatory mechanisms and therapeutic targets .

How can PKN2 antibodies facilitate the investigation of its role in primary cilia formation and regulation?

Recent research has uncovered PKN2's involvement in the negative regulation of ciliogenesis, opening new avenues for investigating its role in ciliopathies and related disorders. PKN2 antibodies can be pivotal in advancing this emerging field:

For studying PKN2's localization during ciliogenesis, co-immunofluorescence using PKN2 antibodies (1:10-1:100) alongside markers for basal bodies (γ-tubulin) and ciliary axonemes (acetylated tubulin) can define its spatial distribution throughout the ciliary cycle. Time-course experiments in serum-starved cells, which induce ciliogenesis, can track temporal changes in PKN2 expression and localization during primary cilia formation and disassembly .

To elucidate molecular mechanisms, researchers can employ proximity ligation assays using PKN2 antibodies together with antibodies against known ciliary regulators to identify novel interactions specific to cilia. Immunoprecipitation of PKN2 (0.5-4.0 μg) from ciliated versus non-ciliated cells, followed by mass spectrometry, can identify context-specific interaction partners. These approaches, complemented by genetic manipulation of PKN2 levels, can reveal how PKN2 contributes to ciliary dynamics. Given the importance of primary cilia in development and disease, these studies may provide insights into developmental disorders and potential therapeutic approaches for ciliopathies .