KIF5B Antibody, HRP conjugated

What is KIF5B and why is it important in cellular research?

KIF5B is a member of the kinesin-1 family of microtubule-based motor proteins. It plays essential roles in intracellular transport processes and has several crucial cellular functions:

• Microtubule-dependent motor required for normal distribution of mitochondria and lysosomes

• Regulates centrosome and nuclear positioning during mitotic entry

• During G2 phase, antagonizes dynein function and drives nuclei and centrosome separation

• Required for anterograde axonal transportation of cargo proteins like MAPK8IP3/JIP3

• Directs lysosome movement toward microtubule plus ends through interactions with PLEKHM2 and ARL8B

• Drives polarization of cytolytic granules and microtubule-organizing centers in NK cell immune synapses

KIF5B contains three major structural domains: a globular N-terminal motor domain responsible for ATP hydrolysis and microtubule binding, a central alpha-helical rod domain involved in dimerization, and a globular C-terminal domain that mediates cargo binding . Its calculated molecular weight is 963 amino acids (110 kDa), though it typically runs at 110-120 kDa on Western blots .

How does KIF5B differ from other KIF5 family members (KIF5A and KIF5C)?

While KIF5A, KIF5B, and KIF5C share structural similarities as kinesin-1 family members, they exhibit several key differences:

• Structural differences:

  • They have distinct C-terminal regions, particularly at the carboxyl-termini, which are important for cargo binding specificity

  • KIF5B has two conserved arginine residues (R941 and R956) that are targets for methylation

• Post-translational modifications:

  • KIF5B and KIF5C, but not KIF5A, undergo arginine methylation

  • Methylation of KIF5B is critical for its interaction with specific cargo proteins like FMRP and G3BP1

• Expression patterns:

  • KIF5B is ubiquitously expressed (hence its alternative name "ubiquitous kinesin heavy chain" or UKHC)

  • KIF5A and KIF5C show more restricted expression patterns, particularly in neurons

• Functional specificity:

  • Despite structural similarities, KIF5B plays non-redundant roles in excitatory synapse development that cannot be compensated by other KIF5 members

These differences highlight the specialized roles of each KIF5 family member and explain why specific antibodies have been developed against unique epitopes for each isoform .

What are the major applications for KIF5B antibodies in research?

KIF5B antibodies are valuable tools in multiple research applications:

These applications have enabled researchers to investigate KIF5B's roles in normal cellular functions and disease states across diverse experimental systems .

How does arginine methylation of KIF5B affect its function in neurons?

Arginine methylation of KIF5B serves as a critical post-translational modification that regulates its function in neurons. Research has revealed several key aspects of this regulation:

• Methylation sites: KIF5B contains two conserved arginine residues (R941 and R956) in its carboxyl-terminus that undergo mono-methylation .

• Detection methods:

  • Antibodies targeting mono-arginine methylation at glycine-rich motifs [MMA (R*GG)]

  • Immunoprecipitation with anti-Mono-Methyl-Arginine antibody followed by KIF5B immunoblotting

• Functional consequences:

  • Protein interactions: Methylation is essential for KIF5B binding to FMRP and G3BP1. Methylation-deficient mutants (R941H/R956H) show significantly reduced pull-down of these interacting proteins .

  • Spine morphogenesis: Wild-type KIF5B, but not methylation-deficient mutants, rescues the loss of mushroom spines induced by KIF5B knockdown in hippocampal neurons .

  • Synaptic transmission: Methylation-deficient mutants fail to reverse the reduction in miniature excitatory postsynaptic current (mEPSC) frequency caused by KIF5B knockdown .

These findings demonstrate that arginine methylation serves as a regulatory mechanism that controls KIF5B's ability to transport specific cargo proteins critical for neuronal development and function .

What are the specific roles of KIF5B in synaptic development and function?

KIF5B plays several crucial roles in synaptic development and function that appear to be distinct from other kinesin family members:

• Dendritic spine morphology:

  • KIF5B is required for the formation and maintenance of mushroom-shaped dendritic spines

  • Knockdown of KIF5B in hippocampal neurons leads to a significant reduction in mushroom spines

  • This function can be rescued by wild-type KIF5B but not by methylation-deficient mutants

• Synaptic transmission:

  • KIF5B depletion reduces the frequency of miniature excitatory postsynaptic currents (mEPSCs)

  • This suggests KIF5B is important for maintaining functional synapses

  • Electrophysiological recordings show that wild-type KIF5B, but not methylation-deficient KIF5B, can rescue the reduction in mEPSC frequency

• Transport of synaptic components:

  • KIF5B mediates the transport of specific cargo proteins including RNA-binding proteins like FMRP and G3BP1

  • These proteins are crucial for local protein synthesis in dendrites and synapses

  • The cargo-binding function depends on post-translational modifications, particularly arginine methylation

These findings highlight KIF5B's non-redundant functions in excitatory synapse development and demonstrate how specific motor proteins contribute to neuronal connectivity and function .

How can researchers distinguish between KIF5B and other KIF5 family members in experimental settings?

Distinguishing between closely related KIF5 family members requires specific approaches:

• Isoform-specific antibodies:

  • Use antibodies generated against unique peptide sequences:

    • KIF5B: antibody against peptide FDKEKANLEAFTADKDIA

    • KIF5A: antibody against peptide NGNATDINDNRSDLPC

    • KIF5C: antibody against peptide SAKDQKSLEPC

• Genetic approaches:

  • KIF5B-specific shRNA for knockdown studies

  • KIF5B knockout cell lines (as demonstrated with HAP1 cells)

  • Isoform-specific PCR primers for expression analysis

• Post-translational modification detection:

  • Use of methylation-specific antibodies can help distinguish KIF5B/C (which undergo methylation) from KIF5A (which does not)

• Antibody validation strategies:

  • Western blot on wild-type versus KIF5B knockout cells

  • Expected molecular weight: 110-120 kDa

  • Validation across multiple applications and sample types

• Functional differentiation:

  • Rescue experiments with specific KIF5 isoforms to determine non-redundant functions

  • Analysis of isoform-specific binding partners

Proper discrimination between KIF5 family members is essential for accurately interpreting experimental results, particularly in tissues where multiple isoforms are expressed.

What are the optimal conditions for using KIF5B antibodies in Western blot applications?

Successful Western blot detection of KIF5B requires careful optimization of several parameters:

It is strongly recommended to titrate the antibody in each experimental system to achieve optimal results, as sensitivity may vary across different sample types and preparation methods .

What are the best practices for using KIF5B antibodies in immunofluorescence and immunohistochemistry?

Optimizing KIF5B detection in immunofluorescence (IF) and immunohistochemistry (IHC) requires attention to several technical details:

Antibody Dilutions:

• For IF/ICC: 1:200-1:800 (21632-1-AP) or 1:50-1:500 (82485-1-RR)

• For IHC: 1:20-1:200 (21632-1-AP) or 1:500-1:2000 (82485-1-RR)

Sample Preparation:

• Validated cell types: HepG2 cells have been specifically validated for IF/ICC

• Validated tissues: Human kidney, skin, and ovarian cancer tissue for IHC

Antigen Retrieval (for IHC):

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

Controls:

• Negative control: KIF5B knockout cells or tissues

• For co-localization studies: Include appropriate markers for cellular compartments

Visualization in Research Applications:

• In neuronal studies, co-transfection with GFP can help visualize cellular morphology alongside KIF5B staining

• For evaluating spine morphology, high-resolution imaging is essential to distinguish spine types

Optimization Notes:

• It is strongly recommended to titrate the antibody for each specific application and sample type

• Image acquisition parameters should be standardized across experimental conditions

• For quantitative analysis, maintain identical exposure settings between samples

Following these guidelines will help ensure specific and reproducible detection of KIF5B across different experimental contexts.

How can researchers validate the specificity of KIF5B antibodies in their experimental systems?

Rigorous validation of KIF5B antibodies is essential for reliable research outcomes. Multiple complementary approaches should be employed:

• Genetic knockout/knockdown validation:

  • Western blot comparison between wild-type and KIF5B knockout cell lysates (example: HAP1 cells)

  • RNA interference using KIF5B-specific shRNA to confirm reduced antibody signal

  • Expected result: Loss or significant reduction of signal at 110-120 kDa

• Multiple antibody approach:

  • Test antibodies targeting different epitopes of KIF5B:

    • Antibodies against amino acids 350-400

    • Antibodies against KIF5B fusion protein Ag16250

  • Consistent results with different antibodies increase confidence in specificity

• Cross-reactivity assessment:

  • Be aware that some antibodies detect both KIF5B and KIF5C (as indicated by "Anti-KIF5B+KIF5C antibody")

  • Use isoform-specific antibodies as needed for discriminating between family members

• Recombinant protein controls:

  • Overexpression of tagged KIF5B constructs as positive controls

  • Competition assays with immunizing peptides

• Application-specific validation:

  • For Western blot: Confirm correct molecular weight (110-120 kDa)

  • For IP: Verify pulled-down protein by mass spectrometry or Western blot

  • For IF/IHC: Compare staining pattern with published localization data

These validation approaches should be documented in publications to enhance reproducibility and reliability of KIF5B-related research findings.

What are common issues when using KIF5B antibodies in Western blot and how can they be resolved?

When working with KIF5B antibodies in Western blot applications, researchers may encounter several challenges:

For optimal troubleshooting, researchers should verify their antibody's expected performance using the manufacturer's validation data and positive controls before conducting critical experiments .

How can researchers troubleshoot issues with immunoprecipitation of KIF5B?

Immunoprecipitation of KIF5B can present specific challenges that require methodical troubleshooting:

• Poor IP efficiency (low yield):

  • Optimize antibody amount: Use 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

  • Verify antibody is validated for IP (Bethyl Laboratories and Proteintech antibodies are specifically validated for IP)

  • Extend incubation time with antibody (overnight at 4°C)

  • Use protein A/G beads appropriate for the antibody host species

  • Pre-clear lysate to reduce non-specific binding

• Co-immunoprecipitation failures:

  • Consider post-translational modifications: Methylation of KIF5B is critical for interaction with partners like FMRP and G3BP1

  • Modify buffer conditions (adjust salt concentration, detergent type/concentration)

  • Use crosslinking to stabilize transient interactions

  • Try different antibody epitopes that don't interfere with protein-protein interaction sites

• High background in IP:

  • Increase washing stringency (more washes or higher salt concentration)

  • Include appropriate negative controls (IgG from same species or KIF5B knockout cells)

  • Pre-block beads with BSA before adding antibody-lysate mixture

• Detection of post-translational modifications:

  • For methylated KIF5B: Use anti-Mono-Methyl-Arginine antibody

  • Consider reciprocal IP approach: IP with modification-specific antibody, then immunoblot for KIF5B

  • Include appropriate inhibitors to preserve modifications

Successful IP of KIF5B has been demonstrated in HepG2 cells and 293T cells (for FLAG-tagged KIF5B constructs) , providing useful reference points for optimization.

How should researchers interpret changes in KIF5B expression or localization in neurons?

Interpreting changes in KIF5B expression or localization in neurons requires careful analysis of multiple parameters:

• Dendritic spine morphology:

  • Reduced KIF5B expression correlates with loss of mushroom spines

  • Interpretation framework: KIF5B is essential for mature spine maintenance

  • Quantification approach: Analyze spine morphology (mushroom, thin, stubby) and density

  • Control comparisons: Compare KIF5B-shRNA to control shRNA; compare rescue with wild-type vs. methylation-deficient KIF5B

• Synaptic transmission:

  • KIF5B knockdown decreases mEPSC frequency but not amplitude

  • Interpretation: KIF5B primarily affects presynaptic release probability or the number of functional synapses rather than postsynaptic receptor density

  • Measurement approach: Analyze frequency, amplitude, and kinetics of miniature excitatory postsynaptic currents

  • Representative data from studies show clear changes in mEPSC traces after KIF5B manipulation

• Subcellular localization:

  • Changes in KIF5B distribution may indicate altered transport dynamics

  • Co-localization with cargo proteins (e.g., FMRP, G3BP1) provides functional insights

  • Altered distribution after stimulation might reflect activity-dependent regulation

• Post-translational modifications:

  • Changes in methylation status affect KIF5B function

  • Detection methods include methylation-specific antibodies

  • Functional consequences can be assessed through rescue experiments with wild-type vs. methylation-deficient constructs

When interpreting such changes, it's important to integrate multiple measurements (morphological, biochemical, and electrophysiological) to develop comprehensive understanding of KIF5B function in neuronal development and plasticity .

How can researchers quantitatively analyze KIF5B expression levels across different experimental conditions?

Rigorous quantitative analysis of KIF5B expression requires systematic approaches across different experimental techniques:

• Western blot quantification:

  • Detection specifications:

    • Expected molecular weight: 110-120 kDa

    • Recommended antibody dilutions: 1:2000-1:16000 or higher based on antibody sensitivity

  • Quantification procedure:

    • Use appropriate loading controls (GAPDH, actin, tubulin)

    • Apply densitometry software to measure band intensity

    • Normalize KIF5B signal to loading control

    • Express as fold change relative to control condition

  • Validation approach:

    • Include KIF5B knockout/knockdown samples as specificity controls

    • Analyze multiple biological replicates (n≥3)

• Immunofluorescence quantification:

  • Image acquisition parameters:

    • Maintain identical exposure settings across all samples

    • Collect z-stacks for three-dimensional analysis when relevant

  • Quantification approaches:

    • Mean fluorescence intensity (whole cell or specific compartments)

    • Distribution pattern analysis

    • Co-localization analysis with cargo markers (Pearson's correlation coefficient)

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution:

    • For normally distributed data: t-test (two conditions) or ANOVA (multiple conditions)

    • For non-normally distributed data: Non-parametric tests like Kruskal-Wallis (as used in )

  • Report means with standard error or deviation

  • Include sample sizes in figure legends and methods

This methodological framework enables reliable quantitative analysis of KIF5B expression, facilitating comparison across experimental conditions and between different studies .