KANK2 Antibody

What is KANK2 and what are its known biological functions?

KANK2 (KN motif and ankyrin repeat domains 2) is a multifunctional protein also known as ANKRD25, KIAA1518, MXRA3, or SIP. It plays several critical roles in cellular function, notably as a key molecule linking integrin αVβ5 adhesion complexes to microtubules, enabling actin-microtubule crosstalk that influences cell migration and sensitivity to microtubule-targeting drugs . KANK2 is involved in multiple cellular processes including:

• Regulation of focal adhesion dynamics and cell migration

• Connection of integrin-based adhesions to the microtubule network

• Formation of actin stress fibers through interaction with ARHGDIA and regulation of Rho signaling

• Transcriptional regulation by sequestering nuclear receptor coactivators (NCOA1, NCOA2, NCOA3) in the cytoplasm

• Regulation of caspase-independent apoptosis by sequestering the proapoptotic factor AIFM1 in mitochondria

• Negative control of vitamin D receptor signaling pathway

• Potential tumor suppressor role in renal cell carcinoma

Understanding these functions is essential when designing experiments with KANK2 antibodies to investigate specific biological questions.

What should researchers consider when selecting a KANK2 antibody for their experiments?

When selecting a KANK2 antibody, researchers should consider several factors to ensure optimal experimental outcomes:

• Target species reactivity: Confirm the antibody recognizes KANK2 in your experimental species. Available antibodies show reactivity with human, mouse, and rat samples .

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF/ICC). For example, antibody 20546-1-AP is validated for IHC and IF/ICC applications , while 21733-1-AP is validated for WB, IHC, and IF/ICC .

• Epitope location: Consider whether the antibody recognizes a specific domain of KANK2 that might be affected by experimental conditions or protein interactions.

• Clonality: Polyclonal antibodies (like those in the search results) offer high sensitivity but potentially lower specificity than monoclonal antibodies.

• Published validation: Check if the antibody has been cited in publications for your intended application .

• Molecular weight detection: The expected molecular weight of KANK2 is approximately 91 kDa, but observed weight in experiments ranges from 95-100 kDa .

Always perform validation experiments with appropriate positive and negative controls to confirm antibody specificity in your experimental system.

What tissues and cell lines show confirmed KANK2 expression for use as positive controls?

Based on available data, researchers should consider the following tissues and cell lines as potential positive controls for KANK2 expression studies:

Positive tissue controls for IHC:

• Human: kidney, colon tissue

• Mouse: heart, ovary, kidney, lung tissue

Positive cell lines for IF/ICC:

• HeLa cells

• HepG2 cells

• A549 cells

Positive samples for Western blot:

• Mouse heart tissue

• Mouse kidney tissue

• Mouse lung tissue

• Transfected HEK-293 cells (for overexpression studies)

The selection of appropriate positive controls is crucial for validating experimental results and confirming antibody specificity. When using these tissues or cell lines, researchers should follow recommended antigen retrieval methods, such as TE buffer (pH 9.0) or citrate buffer (pH 6.0) for IHC applications .

What are the recommended protocols for immunofluorescence localization of KANK2?

For optimal immunofluorescence localization of KANK2, researchers should follow these methodological guidelines based on published protocols:

Sample preparation and fixation:

• For most applications, fix cells with 2% paraformaldehyde for 15-20 minutes at room temperature .

• For co-staining with α-tubulin, methanol fixation is recommended as it better preserves microtubule structures .

• Permeabilize cells with 0.1% Triton X-100 for 5-10 minutes to allow antibody access to intracellular KANK2 .

Antibody incubation and dilution:

• Primary antibody dilution: Use KANK2 antibodies at dilutions between 1:50-1:500 for IF/ICC (21733-1-AP) or 1:200-1:800 (20546-1-AP) .

• Incubate with primary antibody for 1 hour at room temperature or overnight at 4°C .

• Follow with appropriate secondary antibody incubation for 1 hour at room temperature .

Co-staining recommendations:

• For focal adhesion studies, co-stain with markers such as talin, kindlin-2, paxillin, or vinculin .

• For microtubule interaction studies, co-stain with α-tubulin .

• For liprin-β1/ELKS localization studies, use appropriate antibodies against these proteins to visualize the relationship with KANK2 .

Mounting and visualization:

• Mount slides in DAPI Fluoromount-G for nuclear counterstaining .

• Use confocal microscopy for optimal visualization of KANK2 localization patterns, particularly at focal adhesion belts .

When analyzing results, pay particular attention to KANK2 localization at the outer border of mature focal adhesions (the "FA belt") as well as in thin, elongated central adhesions, as these are characteristic KANK2 distribution patterns .

How should researchers optimize Western blot protocols for detecting KANK2?

For optimal Western blot detection of KANK2, researchers should consider the following protocol optimizations:

Sample preparation:

• Lyse cells in a buffer containing protease inhibitors to prevent degradation of KANK2.

• For tissues, homogenize thoroughly in appropriate lysis buffer with protease inhibitors.

• Heat samples in SDS loading buffer at 95°C for 5 minutes before loading.

Gel electrophoresis and transfer:

• Use 8-10% SDS-PAGE gels to effectively resolve the 91-100 kDa KANK2 protein .

• Ensure sufficient separation time to resolve KANK2 from similarly sized proteins.

• Transfer to PVDF or nitrocellulose membrane using standard protocols.

Antibody incubation:

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

• Use KANK2 antibody at recommended dilutions: 1:500-1:3000 for WB (21733-1-AP) .

• Incubate primary antibody overnight at 4°C for optimal sensitivity.

• Wash thoroughly with TBST (3-5 times, 5-10 minutes each).

• Incubate with appropriate HRP-conjugated secondary antibody.

Detection and analysis:

• Expect to observe KANK2 bands at approximately 95-100 kDa .

• Include positive controls such as mouse heart tissue, mouse kidney tissue, or transfected HEK-293 cells .

• For knockdown validation studies, use KANK2-specific siRNA as a negative control .

When troubleshooting, consider that post-translational modifications might affect the apparent molecular weight of KANK2, and that different isoforms might be expressed in different tissues or cell types.

What antigen retrieval methods are recommended for KANK2 immunohistochemistry?

For successful immunohistochemical detection of KANK2 in tissue sections, proper antigen retrieval is critical. Based on the available data, the following methods are recommended:

Primary recommendation:

• Use TE buffer at pH 9.0 for antigen retrieval .

• This method has been validated for detection of KANK2 in human kidney and colon tissue, as well as mouse heart and ovary tissue .

Alternative method:

• Citrate buffer at pH 6.0 can be used as an alternative antigen retrieval method .

• This may be preferable for certain tissue types or when co-staining with other antibodies requiring this retrieval method.

Protocol considerations:

• Heat-induced epitope retrieval (HIER) methods using either pressure cooker or microwave heating are typically effective.

• For TE buffer retrieval, heat slides in buffer for 15-20 minutes followed by cooling to room temperature.

• After retrieval, proceed with standard blocking (3% BSA or serum) before primary antibody incubation.

• Use KANK2 antibody at dilutions between 1:20-1:200 (20546-1-AP) or 1:50-1:500 (21733-1-AP) for IHC applications.

• Develop with appropriate detection system (HRP/DAB or fluorescence-based).

Different tissue types may require optimization of these antigen retrieval conditions for optimal staining results. Always include positive control tissues (human kidney or colon; mouse heart, ovary, or kidney) to validate the retrieval method and antibody performance .

How can researchers accurately visualize and distinguish KANK2 at focal adhesion belts versus other subcellular locations?

Visualizing KANK2 at focal adhesion (FA) belts requires careful experimental design and precise imaging techniques:

Co-localization markers:

• Use multiple focal adhesion markers for co-staining: Talin and kindlin-2 mark the entire FA, while paxillin, ILK, and vinculin show strong signals in the FA core .

• β3 integrins co-localize with paxillin in the core of mature FAs, while total and active β1 integrins accumulate in FA belts along with KANK2 .

• Liprin-β1 and ELKS are useful markers for adjacent regions where KANK2 may also localize .

Imaging and analysis techniques:

• Confocal microscopy with Z-stack acquisition is essential for precise localization studies.

• Perform line profile analysis across focal adhesions to visualize the distribution of KANK2 relative to core FA proteins. KANK2 typically peaks at the outer FA border, where canonical FA proteins show ~50% of their plateau intensity .

• Use fluorescence intensity quantification to measure relative enrichment of KANK2 in different subcellular compartments.

Experimental considerations:

• Culture cells on fibronectin-coated surfaces to promote formation of mature focal adhesions where KANK2 belts are most evident .

• Consider using fibronectin-coated crossbow-shaped micropatterns which enhance the recruitment of KANK2 to FA borders .

• For temporal studies, note that KANK2-positive central adhesions form within 1 hour of plating on fibronectin, before fibronectin fibrillogenesis occurs .

• Blebbistatin treatment can help distinguish nascent adhesions (which lack KANK2) from mature focal adhesions .

By combining these approaches, researchers can accurately distinguish KANK2 at FA belts from other subcellular locations such as central adhesions, stress fibers, or diffuse cytoplasmic distribution.

What experimental approaches can be used to study the functional interaction between KANK2 and the microtubule network?

To investigate the functional interaction between KANK2 and the microtubule network, researchers should consider these advanced experimental approaches:

Co-localization and proximity studies:

• Perform co-immunostaining of KANK2 with α-tubulin using methanol fixation, which preserves microtubule structures .

• Use super-resolution microscopy (STORM, PALM, or SIM) for nanoscale visualization of KANK2-microtubule interactions.

• Apply proximity ligation assays (PLA) to detect direct interaction between KANK2 and microtubule-associated proteins.

Functional perturbation studies:

• Implement KANK2 knockdown using single siRNA (target sequence: ATGTCAACGTGCAAGATGA) and assess changes in microtubule organization and dynamics .

• Express KANK2 deletion constructs lacking specific domains (KN motif, coiled-coil domain, Ank repeats) to determine which domains are essential for microtubule interaction .

• Analyze microtubule plus-end tracking using EB1-GFP in control versus KANK2-depleted cells to assess effects on microtubule dynamics.

Microtubule sensitivity assays:

• Evaluate cellular sensitivity to microtubule poisons (e.g., nocodazole, paclitaxel) in control versus KANK2-knockdown cells .

• Measure microtubule regrowth after cold-induced depolymerization to assess KANK2's role in microtubule stability.

• Analyze microtubule acetylation and detyrosination as markers of microtubule stability in the presence or absence of KANK2.

Cortical microtubule stabilization complex analysis:

• Investigate interactions between KANK2 and other components of the cortical microtubule stabilization complex (liprins α and β, ELKS, LL5β, MACF1, KANK1) .

• Perform co-immunoprecipitation experiments to identify direct binding partners of KANK2 within this complex.

These approaches can provide comprehensive insights into how KANK2 mediates the crosstalk between integrin-based adhesions and the microtubule cytoskeleton, which is critical for cell migration and response to microtubule-targeting agents .

How should researchers design experiments to investigate KANK2's role in cell migration and adhesion?

To effectively investigate KANK2's role in cell migration and adhesion, researchers should design experiments that address both functional outcomes and underlying mechanisms:

Cell migration assays:

• 2D migration: Perform wound healing/scratch assays in control versus KANK2-knockdown cells to quantify migration rates and directionality .

• Transwell migration: Use Boyden chamber assays to assess directed migration toward chemoattractants.

• Single-cell tracking: Implement live-cell imaging with automated tracking to analyze individual cell migration parameters (velocity, persistence, directionality).

• Micropattern-based migration: Utilize micropatterned substrates to study KANK2's role in constrained migration paths.

Adhesion dynamics:

• Focal adhesion turnover: Use FRAP (Fluorescence Recovery After Photobleaching) of tagged adhesion proteins to measure turnover rates in the presence or absence of KANK2.

• Traction force microscopy: Quantify cellular force generation on deformable substrates to assess how KANK2 influences cell-substrate mechanical interactions.

• Adhesion size and maturation: Analyze focal adhesion size, number, and distribution using immunofluorescence of adhesion markers (paxillin, vinculin) in control versus KANK2-manipulated cells.

Fibronectin fibrillogenesis assessment:

• Fibronectin matrix assembly: Evaluate FN fibril formation using immunofluorescence staining in cells expressing FL-KANK2-GFP versus GFP alone in KANK2-depleted cells .

• Time-course analysis: Monitor FN fibrillogenesis at multiple time points (1h, 5h, overnight) to capture the temporal progression of matrix assembly .

Molecular mechanism investigations:

• Domain-specific functions: Express KANK2 deletion constructs (ΔKNΔ, Δcoil, Δ(1-670)) to determine which domains are essential for migration and adhesion .

• Interaction with Rho signaling: Analyze Rho GTPase activity using FRET-based biosensors in control versus KANK2-knockdown cells .

• Integrin specificity: Investigate whether KANK2's effects are specific to certain integrin heterodimers (e.g., αVβ5, β1 integrins) .

When designing these experiments, researchers should consider using multiple cell types, as KANK2's role might vary between different cellular contexts. Additionally, complementary approaches (gain- and loss-of-function) provide more robust insights into KANK2's functional roles.

What are common challenges in KANK2 detection and how can they be addressed?

Researchers working with KANK2 antibodies may encounter several challenges. Here are common issues and their solutions:

Weak or absent KANK2 signal in immunostaining:

• Issue: Insufficient antigen retrieval or fixation method incompatibility

• Solution: Compare paraformaldehyde (standard) versus methanol fixation (better for co-staining with α-tubulin) . Optimize antigen retrieval using either TE buffer (pH 9.0) or citrate buffer (pH 6.0) .

• Issue: Antibody concentration too low

• Solution: Titrate antibody concentrations within recommended ranges (1:50-1:500 for IHC; 1:200-1:800 for IF/ICC) .

• Issue: Low KANK2 expression in sample

• Solution: Use known positive controls (human kidney/colon tissue, mouse heart/ovary tissue, HeLa cells) . Consider transfecting cells with KANK2 expression constructs as positive controls.

Nonspecific background in immunostaining:

• Issue: Insufficient blocking or antibody cross-reactivity

• Solution: Extend blocking time (1-2 hours), increase blocking agent concentration (5% BSA or serum), and include additional washing steps. Validate specificity using KANK2 knockdown controls .

Multiple bands in Western blot:

• Issue: Protein degradation or detection of multiple isoforms

• Solution: Use fresh samples with complete protease inhibitors. KANK2's expected molecular weight is 91 kDa, but it may run at 95-100 kDa . Confirm specificity using KANK2 knockdown or knockout samples.

Difficulty visualizing FA belt localization:

• Issue: Immature focal adhesions or inappropriate cell culture conditions

• Solution: Culture cells on fibronectin-coated surfaces for at least 24-48 hours to allow maturation of focal adhesions . Consider using crossbow-shaped micropatterns which enhance KANK2 recruitment to FA borders .

Inconsistent results between experiments:

• Issue: Variability in KANK2 expression or localization

• Solution: Standardize cell culture conditions, passage number, and cell density. Include internal controls in each experiment for normalization.

By addressing these common challenges systematically, researchers can improve the reliability and reproducibility of their KANK2 studies.

How can researchers accurately distinguish between KANK1 and KANK2 in their experiments?

Distinguishing between KANK1 and KANK2 is critical for accurate experimental interpretation, as both proteins share structural similarities and may have overlapping functions:

Antibody selection and validation:

• Use antibodies raised against unique regions of KANK2 that don't cross-react with KANK1.

• Validate antibody specificity using overexpression and knockdown controls for both KANK1 and KANK2.

• Perform Western blot analysis to confirm the antibody detects a single band of the expected size for KANK2 (91-100 kDa) .

mRNA expression analysis:

• Design PCR primers or probes that target unique regions of KANK2 mRNA.

• Use quantitative RT-PCR with high-specificity primers to distinguish between KANK1 and KANK2 expression levels.

• Consider RNA-seq analysis for comprehensive profiling of all KANK family members.

Localization pattern differences:

• While both KANK1 and KANK2 localize to adhesion structures, careful analysis of their distribution patterns may reveal subtle differences.

• KANK2 specifically localizes to FA belts and central adhesions, with characteristic puncta at the outer FA border .

• Perform co-immunostaining of KANK1 and KANK2 (using differently labeled secondary antibodies) to directly compare their localization.

Functional analysis:

• Design specific siRNA sequences that target KANK2 without affecting KANK1 expression (e.g., target sequence: ATGTCAACGTGCAAGATGA) .

• Perform rescue experiments with KANK2 constructs resistant to siRNA to confirm phenotype specificity.

• Compare phenotypes of KANK1 versus KANK2 knockdown to identify distinct functions.

Domain-specific approaches:

• Utilize the differences in domain structures between KANK proteins for specific detection or functional studies.

• Express domain-specific constructs (e.g., KN motif only, ankyrin repeats only) to study isoform-specific functions .

By employing these approaches, researchers can confidently distinguish between KANK1 and KANK2 in their experimental systems and accurately attribute observed phenotypes to the correct protein.

What considerations should be made when interpreting KANK2 localization in different cell types and tissue contexts?

When interpreting KANK2 localization across different biological contexts, researchers should consider several important factors:

Cell type-specific variations:

• KANK2 expression levels vary significantly between cell types, with notable expression in kidney, heart, colon, ovary, and lung tissues .

• Cell-type specific adhesion structures may influence KANK2 localization patterns. For example, highly migratory cells versus stationary epithelial cells may show different distribution of KANK2 at adhesion sites.

• The composition of the extracellular matrix can affect KANK2 localization. Fibronectin-rich environments promote KANK2 localization to FA belts and involvement in fibronectin fibrillogenesis .

Developmental and disease contexts:

• KANK2 may play specific roles in developing tissues, such as regulating podocyte migration during kidney development .

• In pathological contexts like renal cell carcinoma, KANK2 has been identified as a potential tumor suppressor , suggesting its expression and localization may be altered in cancer cells.

• Consider how tissue microenvironment and cellular stress might affect KANK2 localization and function.

Technical considerations for tissue sections:

• Tissue fixation and processing methods significantly impact antigen preservation and antibody accessibility.

• For KANK2 IHC in tissues, antigen retrieval using TE buffer (pH 9.0) is recommended, with citrate buffer (pH 6.0) as an alternative .

• Tissue architecture can complicate interpretation of KANK2 localization; use multiple tissue sections and controls for accurate assessment.

Integrin-subtype dependent localization:

• KANK2 shows specific association with integrin αVβ5 adhesion complexes , while active β1 integrins accumulate in FA belts along with KANK2 .

• The integrin expression profile of different cell types may therefore influence KANK2 localization patterns.

Interpreting co-localization data:

• Use multiple markers to define subcellular compartments (FA core, FA belt, stress fibers, etc.).

• Quantitative co-localization analysis (Pearson's coefficient, Manders' overlap) can provide objective measures of spatial association between KANK2 and other proteins.

• Consider three-dimensional analysis in tissue sections rather than relying solely on single optical sections.

By considering these various factors, researchers can more accurately interpret KANK2 localization data across different experimental systems and biological contexts.

What are promising research areas for further understanding KANK2 function in normal physiology and disease?

Several promising research directions emerge from current understanding of KANK2 biology that warrant further investigation:

KANK2 in cancer biology:

• Explore KANK2's potential role as a tumor suppressor, particularly in renal cell carcinoma .

• Investigate how KANK2-mediated regulation of cell migration contributes to cancer invasion and metastasis .

• Study the relationship between KANK2 expression and sensitivity to microtubule-targeting chemotherapeutics, which could inform personalized treatment approaches .

KANK2 in mechanotransduction:

• Examine how KANK2 participates in cellular responses to mechanical forces through its connections to both focal adhesions and the microtubule network.

• Investigate whether KANK2 is involved in mechanosensitive gene expression through its role in sequestering transcriptional coactivators .

• Study KANK2's function in tissues under high mechanical stress, such as cardiac muscle, where it shows significant expression .

Developmental biology:

• Explore KANK2's role in kidney development, particularly in podocyte migration and glomerular formation .

• Investigate KANK2 function during embryonic development in tissues where it shows high expression (heart, kidney, lung) .

• Study potential compensatory mechanisms between KANK family members during development.

KANK2 in specialized cell functions:

• Examine KANK2's role in immune cell migration and function.

• Investigate KANK2's contribution to neurite outgrowth and neuronal migration, given the importance of microtubule-adhesion crosstalk in these processes.

• Study how KANK2 affects specialized adhesion structures in epithelial cells (tight junctions, adherens junctions) versus classical focal adhesions.

Therapeutic targeting:

• Explore the therapeutic potential of modulating KANK2 function in diseases characterized by dysregulated cell migration.

• Investigate whether KANK2 status could serve as a biomarker for response to microtubule-targeting agents in cancer therapy .

• Consider KANK2's role in fibrosis through its involvement in fibronectin fibrillogenesis and matrix remodeling .

These research directions could significantly advance our understanding of KANK2 biology and potentially reveal new therapeutic opportunities for diseases involving dysregulated cell adhesion, migration, or microtubule dynamics.

How can advanced imaging and molecular techniques enhance the study of KANK2 localization and dynamics?

Advanced technologies offer exciting opportunities to deepen our understanding of KANK2 biology:

Super-resolution microscopy:

• STORM/PALM: These techniques can resolve KANK2 localization at nanoscale resolution (~20nm), allowing precise mapping of KANK2 relative to focal adhesion components and microtubules .

• SIM (Structured Illumination Microscopy): Provides ~100nm resolution with standard fluorophores, enabling detailed visualization of KANK2 at FA belts.

• STED microscopy: Offers live-cell super-resolution imaging to track KANK2 dynamics during adhesion maturation.

Live-cell imaging approaches:

• FRAP (Fluorescence Recovery After Photobleaching): Measure KANK2 turnover rates at FA belts versus other subcellular locations.

• Photoactivatable/photoconvertible KANK2: Track KANK2 molecule fate after activation at specific subcellular locations.

• TIRF microscopy: Visualize KANK2 dynamics specifically at the cell-substrate interface with high signal-to-noise ratio.

Proximity-based protein interaction methods:

• BioID or TurboID: Identify proteins in proximity to KANK2 in living cells by fusing KANK2 to a promiscuous biotin ligase.

• APEX2 proximity labeling: Map the KANK2 interactome with subcellular resolution.

• PLA (Proximity Ligation Assay): Visualize and quantify direct interactions between KANK2 and suspected binding partners in situ.

Optogenetic and acute manipulation:

• Optogenetic recruitment/displacement: Use light-controlled systems to acutely recruit or displace KANK2 from specific subcellular locations.

• Acute protein degradation: Apply degron-based approaches to rapidly deplete KANK2 and observe immediate consequences.

• Domain-specific inhibition: Develop tools to acutely disrupt specific KANK2 domain functions without affecting the whole protein.

Genome engineering and high-content screening:

• CRISPR-Cas9 knock-in: Generate endogenously tagged KANK2 to visualize native protein dynamics without overexpression artifacts.

• Domain-specific CRISPR editing: Create precise mutations in specific KANK2 domains to dissect their functions.

• High-content imaging screens: Identify regulators of KANK2 localization or function through large-scale screening approaches.

These advanced techniques, used individually or in combination, can provide unprecedented insights into KANK2 biology, revealing dynamic behaviors and molecular interactions that are not apparent with conventional approaches.