KIF13B Antibody

What is KIF13B and what are its main structural features?

KIF13B (also known as GAKIN or kinesin-like protein KIF13B) is a 1,826 amino acid microtubule-dependent motor protein that facilitates intracellular transport. Its structure includes three characteristic domains: an N-terminal motor domain containing an ATP-binding motif that powers movement along microtubules, a Forkhead-associated (FHA) domain that binds diverse cargo, and a large stalk domain that mediates protein-protein interactions. Additionally, KIF13B contains a highly conserved CAP-Gly domain at its C-terminus that is essential for microtubule interaction. The protein is primarily localized in the cytoplasm and associated with the cytoskeleton, with highest expression in brain and kidney tissues.

What applications are KIF13B antibodies suitable for in research?

KIF13B antibodies are suitable for multiple research applications including Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and immunoprecipitation (IP). These applications allow researchers to detect, quantify, localize, and isolate KIF13B in various experimental systems. For instance, Western blotting can confirm KIF13B knockdown efficiency, while immunoprecipitation can be used to study KIF13B interactions with binding partners such as VEGFR2. Immunohistochemistry is valuable for examining KIF13B localization within tissues or cellular compartments, particularly in relation to the Golgi apparatus and microtubule structures.

How should researchers select the appropriate KIF13B antibody for their specific research needs?

When selecting a KIF13B antibody, researchers should consider several critical factors: (1) Species reactivity - ensure the antibody recognizes KIF13B in your model system (human, mouse, rat, etc.). (2) Application compatibility - verify the antibody is validated for your intended application (WB, IHC, IP, ELISA). (3) Epitope specificity - some antibodies target specific regions (e.g., N-terminal region) which may be important depending on your research question. (4) Clonality - monoclonal antibodies like the 6E11 clone offer high specificity for particular epitopes, while polyclonal antibodies might provide broader recognition. (5) Published validation - check whether the antibody has been cited in peer-reviewed publications. For example, if studying KIF13B interactions with VEGFR2, an antibody validated for immunoprecipitation would be essential.

What are the optimal conditions for detecting KIF13B using Western blotting?

For optimal Western blot detection of KIF13B (202.8 kDa), researchers should implement specific technical considerations: (1) Use low percentage (6-8%) polyacrylamide gels to properly resolve this high molecular weight protein. (2) Extend transfer time (possibly overnight at low voltage) to ensure complete transfer of this large protein. (3) Block with 5% non-fat milk or BSA in TBST for 1-2 hours at room temperature. (4) Incubate with primary KIF13B antibody (typically at 1:500-1:1000 dilution) overnight at 4°C. (5) Following TBST washes, incubate with appropriate HRP-conjugated secondary antibody. (6) For enhanced sensitivity, consider using chemiluminescent substrates specifically designed for high molecular weight proteins. (7) When analyzing tissues, brain and kidney samples will typically show the highest expression levels. (8) Include positive controls from tissues known to express KIF13B and negative controls from KIF13B-knockdown cells to validate specificity.

How can researchers effectively design KIF13B knockdown experiments to study its functional roles?

To design effective KIF13B knockdown experiments, researchers should follow these methodological steps: (1) Select appropriate targeting sequences for shRNA or siRNA design, ensuring they specifically target KIF13B without affecting closely related proteins like KIF13A. (2) Use lentiviral delivery systems for stable knockdown in difficult-to-transfect cells like endothelial cells. (3) Validate knockdown efficiency by both RT-PCR and Western blot, with the latter confirming reduced protein levels. (4) Include appropriate controls including scrambled shRNA sequences. (5) Confirm specificity by testing for unchanged expression of KIF13A and other structurally related kinesins. (6) For functional analyses, incorporate phenotypic assays relevant to KIF13B function, such as endothelial cell sprouting assays, cell migration assays, or angiogenesis models. (7) For rescue experiments, express shRNA-resistant KIF13B constructs to confirm phenotype specificity. This approach has been successfully used to demonstrate KIF13B's specific role in VEGFR2 trafficking and angiogenesis.

What experimental approaches can be used to study KIF13B-mediated protein trafficking?

To investigate KIF13B-mediated protein trafficking, researchers can employ several complementary experimental approaches: (1) Optiprep gradient separation to isolate and analyze subcellular fractions (particularly Golgi, plasma membrane, and endosomal fractions) and assess the distribution of KIF13B and its cargo proteins like VEGFR2. (2) Co-immunoprecipitation assays to detect physical interactions between KIF13B and cargo proteins, tracking the kinetics of these interactions after stimulation (e.g., with VEGF). (3) Live-cell imaging using fluorescently tagged cargo proteins (e.g., VEGFR2-GFP) in combination with KIF13B knockdown to visualize trafficking defects, quantifying parameters like vesicle velocity and transport distance using kymograph analysis. (4) Surface biotinylation assays to quantify the plasma membrane localization of cargo proteins in control versus KIF13B-depleted cells. (5) Immunofluorescence microscopy with markers for specific cellular compartments (e.g., GM130 for Golgi, LAMP2 for lysosomes) to track cargo protein localization. These approaches have revealed that KIF13B interacts directly with VEGFR2 primarily at the Golgi and facilitates its transport to the cell surface after VEGF stimulation.

How does KIF13B regulate VEGFR2 trafficking and angiogenesis?

KIF13B plays a critical regulatory role in angiogenesis through its function as a motor protein that delivers VEGFR2 from the Golgi to the plasma membrane. Mechanistically, KIF13B interacts directly with VEGFR2 on microtubules, with this interaction increasing up to 5.5-fold within 1 hour of VEGF stimulation and remaining elevated for up to 4 hours. Subcellular fractionation studies demonstrate that KIF13B is primarily localized to the Golgi fraction alongside VEGFR2, suggesting this as the primary site of cargo loading. Following VEGF-induced receptor internalization, KIF13B mediates the restoration of VEGFR2 surface expression by transporting newly synthesized receptors from the Golgi to the plasma membrane. This transport is essential for sustained VEGFR2 activation and downstream signaling. Functionally, KIF13B depletion impairs endothelial cell migration, tube formation, and neovascularization in mouse models. These findings illustrate that KIF13B-mediated trafficking is a rate-limiting step in the angiogenic process, positioning KIF13B as a potential therapeutic target for angiogenesis-related disorders.

What methods can be used to visualize and quantify KIF13B-mediated vesicular transport in real-time?

To visualize and quantify KIF13B-mediated vesicular transport in real-time, researchers should implement the following methodological approach: (1) Express fluorescently tagged cargo proteins (e.g., VEGFR2-GFP) in control and KIF13B-depleted cells. (2) Utilize spinning disk confocal microscopy with environmental control (37°C, 5% CO2) for live-cell imaging at high temporal resolution (e.g., 5 frames/second) to capture fast vesicular movements. (3) Apply specific stimuli (e.g., VEGF) to initiate transport events and continue imaging for extended periods (30+ minutes) to capture the full trafficking response. (4) Generate kymographs (space-time plots) from linear regions of interest to visualize movement trajectories. (5) Quantify multiple parameters including vesicle velocity (μm/second), distance moved (μm), directionality (anterograde vs. retrograde), and pause frequency. (6) For more sophisticated analysis, implement particle tracking algorithms to automatically detect and follow hundreds of vesicles simultaneously. (7) Co-express fluorescently tagged microtubule markers (e.g., EB1-mCherry) to correlate vesicle movement with microtubule dynamics. This approach has demonstrated that VEGF stimulation activates both fast and long-range transport of VEGFR2 vesicles in control cells, while KIF13B depletion significantly impairs this movement.

What experimental models are most appropriate for studying KIF13B function in angiogenesis?

To comprehensively investigate KIF13B function in angiogenesis, researchers should consider implementing a multiscale approach with these complementary models: (1) 3D endothelial spheroid sprouting assays, where endothelial cells are cultured on microcarrier beads embedded in fibrin gels, allowing quantification of sprout length and branching complexity in KIF13B-depleted versus control cells. (2) In vitro tube formation assays on Matrigel to assess the capacity of endothelial cells to form capillary-like networks. (3) Scratch wound migration assays to evaluate endothelial cell motility. (4) Ex vivo aortic ring assays, where mouse aortic segments are embedded in matrix and sprouting is measured, bridging in vitro and in vivo approaches. (5) In vivo Matrigel plug assays in mice with endothelial-specific KIF13B knockdown to assess neovascularization capacity. (6) Developmental models such as zebrafish or mouse retinal angiogenesis to evaluate KIF13B function in a physiological context. (7) Tumor xenograft models to study the role of KIF13B in pathological angiogenesis. These models, progressing from simple to complex, allow researchers to dissect specific aspects of KIF13B function while confirming physiological relevance.

What are the common pitfalls in KIF13B detection and how can researchers overcome them?

Researchers face several challenges when detecting KIF13B (202.8 kDa) that can be addressed with specific technical solutions: (1) Poor Western blot detection - use fresh lysates prepared with phosphatase and protease inhibitors, optimize gel percentage (6-8%), extend transfer time, and consider specialized transfer systems for high molecular weight proteins. (2) Background or non-specific bands - increase blocking time, optimize antibody dilution, and include knockout/knockdown controls to identify specific bands. (3) Cross-reactivity with KIF13A (the closest homolog) - select antibodies specifically validated against both proteins, and always include KIF13A expression analysis as a specificity control. (4) Inconsistent immunoprecipitation results - pre-clear lysates, optimize antibody concentration, and consider using magnetic beads coated with protein A/G for more efficient capture. (5) Poor signal in immunofluorescence - test different fixation methods (paraformaldehyde versus methanol), optimize permeabilization conditions, and use antigen retrieval if necessary. (6) Variable expression across cell types - normalize to housekeeping proteins and be aware that KIF13B expression is highest in brain and kidney tissues.

How should researchers interpret discrepancies between KIF13B protein levels and functional outcomes in knockdown experiments?

When interpreting discrepancies between KIF13B protein levels and functional outcomes in knockdown experiments, researchers should consider these methodological factors: (1) Threshold effects - KIF13B may require only a minimal amount for function, so partial knockdown might not yield proportional functional effects. (2) Compensation by related proteins - other kinesin family members (particularly KIF13A) may partially compensate for KIF13B loss, requiring evaluation of these related proteins. (3) Cell type-specific dependencies - some cells may be more reliant on KIF13B than others based on expression of accessory or redundant proteins. (4) Cargo-specific effects - KIF13B transports multiple cargoes, so knockdown might affect some trafficking pathways more than others. (5) Timing considerations - acute versus chronic depletion may yield different results due to cellular adaptation. (6) Stimulation-dependent functions - as seen with VEGF stimulation, KIF13B function may only become crucial under specific signaling conditions. (7) Technical verification - confirm knockdown at both mRNA and protein levels, and verify that control shRNAs don't affect KIF13B or related proteins. When facing discrepancies, researchers should examine multiple functional readouts (e.g., both receptor localization and downstream signaling) to build a more complete understanding of KIF13B's role.

What controls should be included when studying KIF13B-mediated protein trafficking to distinguish from general trafficking defects?

To distinguish KIF13B-specific trafficking defects from general trafficking disruption, researchers should implement these essential controls: (1) Analyze multiple cargo proteins, including those known to be KIF13B-dependent (e.g., VEGFR2) and those transported by other mechanisms to verify specificity. (2) Examine the integrity of trafficking compartments (Golgi, endosomes, lysosomes) using specific markers (GM130, EEA1, LAMP2) to ensure general organelle architecture is maintained. (3) Assess microtubule dynamics and organization with markers like EB1-GFP and antibodies against acetylated and tyrosinated tubulin to confirm that KIF13B depletion does not affect the cytoskeletal "highways." (4) Include rescue experiments expressing wild-type KIF13B or specific domains in knockdown cells to restore function. (5) Compare motor-dead KIF13B mutants (ATP-binding site mutations) with wild-type expression to confirm motor activity dependence. (6) Evaluate protein synthesis using techniques like RT-PCR to distinguish trafficking defects from synthesis problems. (7) Examine multiple timepoints after stimulation (e.g., VEGF treatment) to capture the full trafficking response. These controls have confirmed that KIF13B knockdown specifically impairs VEGFR2 trafficking without affecting microtubule dynamics or general trafficking machinery.

How can domain-specific antibodies against KIF13B be used to dissect its molecular functions?

Domain-specific antibodies against KIF13B offer powerful tools for dissecting its molecular functions through these methodological approaches: (1) Epitope mapping studies - antibodies targeting different domains (motor, FHA, stalk, CAP-Gly) can reveal which regions are accessible in various cellular contexts or protein complexes. (2) Functional blocking experiments - antibodies that recognize functional domains can be microinjected to acutely inhibit specific activities without altering protein levels. (3) Conformational state detection - conformation-specific antibodies can distinguish between active (ATP-bound) and inactive states of the motor domain. (4) Protein complex analysis - domain-specific antibodies used in immunoprecipitation can reveal which regions mediate interactions with different binding partners. (5) Post-translational modification detection - phospho-specific antibodies can monitor regulatory modifications that control KIF13B activity. (6) Super-resolution microscopy - domain-specific antibodies with appropriate fluorophores can reveal the nanoscale organization of KIF13B relative to microtubules and cargo. (7) Proximity ligation assays - pairs of antibodies recognizing KIF13B domains and potential interacting proteins can confirm close associations in situ. These approaches can help determine which KIF13B domains are essential for specific functions, such as the direct interaction with VEGFR2 during trafficking.

What is the relationship between KIF13B dysfunction and disease pathologies?

KIF13B dysfunction has been implicated in several disease pathologies through its critical role in intracellular transport mechanisms. While not extensively detailed in the provided search results, we can infer from the molecular functions that KIF13B disruption would impact several physiological systems: (1) Vascular disorders - given KIF13B's essential role in VEGFR2 trafficking and angiogenesis, dysfunction could contribute to pathological angiogenesis in cancer or insufficient angiogenesis in ischemic diseases. (2) Neurological disorders - with high expression in the brain and its role in transport along microtubules, KIF13B defects might impact neuronal function and contribute to neurodegenerative conditions. (3) Developmental abnormalities - proper trafficking of signaling receptors is crucial during development, suggesting KIF13B dysfunction could impact embryonic development. (4) Cancer progression - altered receptor trafficking can dysregulate growth factor signaling pathways implicated in tumorigenesis and metastasis. (5) Kidney pathologies - given high KIF13B expression in kidney tissue, dysfunction might contribute to renal disorders. Research into these relationships would benefit from tissue-specific conditional knockout models and human genetic studies correlating KIF13B variants with disease phenotypes.

How might comparative studies of KIF13B and KIF13A advance our understanding of kinesin specificity in trafficking pathways?

Comparative studies of KIF13B and its closest homolog KIF13A would provide valuable insights into kinesin specificity through these research strategies: (1) Generate parallel knockdown or knockout models of each kinesin individually and in combination to identify unique versus redundant functions. (2) Perform detailed cargo identification using proximity labeling techniques coupled with mass spectrometry for each kinesin to catalog their specific and shared cargoes. (3) Conduct domain-swapping experiments where motor, cargo-binding, or regulatory domains are exchanged between KIF13A and KIF13B to determine which regions confer cargo specificity. (4) Analyze tissue-specific expression patterns to identify contexts where one kinesin may compensate for the other. (5) Examine regulatory mechanisms including phosphorylation sites and binding partners that might differentially control these kinesins. (6) Investigate structural determinants of microtubule interaction, as differences in the CAP-Gly domains might influence microtubule preference or processivity. (7) Compare the roles of these kinesins in different trafficking pathways (e.g., endosomal recycling versus Golgi-to-plasma membrane transport). This comparative approach would reveal the molecular basis for functional specificity in the kinesin family and potentially identify contexts where therapeutic targeting of one kinesin would yield specific effects without disrupting general transport mechanisms.