KIF5B Antibody

What is KIF5B and why is it important for cellular function?

KIF5B is a member of the kinesin-1 family of motor proteins that plays essential roles in intracellular transport. It contains three structural domains: a globular N-terminal motor domain, a central alpha-helical rod domain, and a globular C-terminal domain . KIF5B is ubiquitously expressed, whereas its family members KIF5A and KIF5C are specifically expressed in neurons . KIF5B functions as a microtubule-dependent motor required for normal distribution of mitochondria and lysosomes . It regulates centrosome and nuclear positioning during mitotic entry and, during the G2 phase of the cell cycle, antagonizes dynein function to drive the separation of nuclei and centrosomes in a BICD2-dependent manner . The protein is essential for embryonic development, as targeted disruption of the KIF5B gene results in embryonic lethality .

What is the molecular weight of KIF5B and how can I confirm antibody specificity?

KIF5B has a calculated molecular weight of 110 kDa (963 amino acids), with observed molecular weights typically between 110-120 kDa on Western blots . When validating a KIF5B antibody, look for a single band at this molecular weight. Specificity can be confirmed by using KIF5B knockout or knockdown samples as negative controls . When testing cross-reactivity with other KIF5 family members, recombinant KIF5A, KIF5B, and KIF5C proteins can be used. Some antibodies might show cross-reactivity; for instance, in one study, an antibody against the N-terminal of KIF5A (5A-n) showed slight cross-reactivity with KIF5B, while an antibody against the C-terminal of KIF5C (5C-c) cross-reacted with both KIF5A and KIF5B .

How do KIF5B, KIF5A, and KIF5C differ functionally?

Though structurally similar, these kinesin family members have distinct expression patterns and non-redundant functions:

• KIF5B: Ubiquitously expressed; essential for embryonic development and organelle distribution

• KIF5A and KIF5C: Specifically expressed in neurons

Post-translational modifications also differ. KIF5B and KIF5C undergo arginine methylation at their glycine-rich motifs, while KIF5A does not . This suggests different regulatory mechanisms for these motor proteins. Functionally, these proteins cannot fully compensate for one another. Studies have shown that homologous motor proteins of the kinesin-1 family have non-redundant functions in regulating development , emphasizing the importance of using specific antibodies when studying individual kinesin family members.

What applications are KIF5B antibodies validated for?

KIF5B antibodies have been validated for multiple applications across different sample types:

Most antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 and should be stored at -20°C . It is always recommended to titrate the antibody in each specific testing system to obtain optimal results .

How should I optimize Western blotting protocols for KIF5B detection?

For optimal KIF5B detection by Western blotting:

• Sample preparation: KIF5B is expressed in various cell types. A549, HeLa, HepG2, Jurkat cells, and mouse/rat brain and heart tissues have all been successfully used .

• Antibody dilution: Start with the manufacturer's recommendation (typically between 1:1000 to 1:50000) . Cell Signaling Technology's KIF5B antibody (#18148) has been validated at 1:1000 dilution .

• Expected band size: Look for a single band at 110-120 kDa .

• Controls: Include positive controls (cells/tissues known to express KIF5B) and, if available, KIF5B knockout/knockdown samples as negative controls .

• Specificity verification: If investigating specific KIF5 family members, consider running recombinant KIF5A, KIF5B, and KIF5C proteins to check for cross-reactivity .

What are the key considerations for successful immunofluorescence with KIF5B antibodies?

For immunofluorescence studies of KIF5B:

• Fixation and permeabilization: Standard protocols using paraformaldehyde fixation work well for KIF5B detection .

• Antibody dilution: Use recommended dilutions (typically 1:50-1:800) .

• Imaging parameters: For confocal microscopy, successful imaging has been performed using:

  • 60× (NA 1.4) oil immersion objective

  • For GFP-tagged constructs: 488 nm excitation at 5% transmission, emission collected at 500–530 nm

  • For Alexa 594: 543 nm excitation at 25% transmission, emission collected at 555–625 nm

  • For Alexa 647: 633 nm excitation at 5% transmission, emission collected at >650 nm

  • Images acquired at 512 × 512 pixel resolution

• Image analysis: ImageJ software has been successfully used to analyze KIF5B localization and expression levels .

How can I use KIF5B antibodies to study its role in vesicular transport?

KIF5B plays critical roles in vesicular transport, particularly for Rab6-positive secretory vesicles. To investigate this function:

• Co-localization studies: Use KIF5B antibodies together with markers for vesicles (e.g., Rab6) to assess co-localization by immunofluorescence .

• Functional assays: Compare vesicle transport in control versus KIF5B knockout cells:

  • KIF5B knockout significantly reduces the number of moving vesicles

  • The few remaining moving vesicles in KIF5B knockout cells show significantly higher speeds (exceeding 3 μm/s) compared to control cells

• Rescue experiments: Re-express KIF5B in knockout cells to confirm specificity:

  • Expression of KIF5B in KIF5B knockout cells restores vesicle runs to control values

  • This approach helps distinguish the roles of different kinesin family members

• Analysis of cargo-specific effects: KIF5B is involved in the transport of specific cargoes, such as the Kv1.5 cardiac potassium channel. In H9c2 myoblasts and HEK293 cells, expression of dominant-negative KIF5B (Kif5bDN) prevents surface expression of Kv1.5 despite robust internal expression of the channel .

What methodological approaches can I use to investigate KIF5B's role in neuronal function?

Despite being ubiquitously expressed, KIF5B plays crucial roles in neuronal function:

• Conditional knockout models: KIF5B conditional knockout mice exhibit deficits in:

  • Dendritic spine morphogenesis

  • Synaptic plasticity

  • Memory formation

• Arginine methylation analysis: KIF5B undergoes arginine methylation at conserved residues (R941 and R956):

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

  • Methylation-deficient mutants (R941H/R956H) can be created to study the functional significance of this modification

• Analysis of KIF5B interaction with neuronal proteins: KIF5B's interactions with proteins like FMRP (Fragile X Mental Retardation Protein) can be studied through co-immunoprecipitation followed by Western blotting .

• Synaptic function assays: Compare synaptic protein distribution and electrophysiological properties between wild-type and KIF5B-deficient neurons to understand its role in synaptic function .

How can KIF5B antibodies be used to investigate its roles in mitosis and cell division?

KIF5B regulates centrosome and nuclear positioning during mitotic entry:

• Cell cycle-specific analysis: Synchronize cells at specific cell cycle stages and analyze KIF5B localization and function using immunofluorescence .

• Co-localization with centrosomes and nuclei: Investigate KIF5B's role in nuclear-centrosome separation during G2 phase through co-staining with nuclear and centrosomal markers .

• Interaction with BICD2: Study how KIF5B interacts with BICD2 to antagonize dynein function during nuclear-centrosome separation using co-immunoprecipitation and immunofluorescence .

• Live cell imaging: Use fluorescently tagged KIF5B constructs combined with fixed-cell antibody staining to track KIF5B dynamics during cell division .

How do I distinguish between specific and non-specific signals when using KIF5B antibodies?

To ensure specificity when using KIF5B antibodies:

• Include appropriate controls:

  • Positive controls: Samples known to express KIF5B (e.g., HeLa, HepG2, A549 cells)

  • Negative controls: KIF5B knockout/knockdown samples, secondary antibody-only controls

• Verify band size: KIF5B should appear at 110-120 kDa on Western blots .

• Cross-validate with multiple antibodies: Use antibodies targeting different epitopes of KIF5B to confirm findings .

• Peptide competition assays: Pre-incubating the antibody with the immunizing peptide should eliminate specific signals.

• Use family member-specific antibodies: For experiments requiring discrimination between KIF5A, KIF5B, and KIF5C, use antibodies targeting low-homology regions, such as:

  • N-terminal junction regions (e.g., antibodies designated as 5A-n, 5B-n, 5C-n)

  • C-terminal regions (e.g., antibodies designated as 5A-c, 5B-c)

What are common pitfalls when working with KIF5B antibodies and how can I avoid them?

Common pitfalls and solutions include:

• Cross-reactivity with other KIF5 family members:

  • Solution: Use highly specific antibodies targeting low-homology regions

  • Validate specificity using recombinant KIF5A, KIF5B, and KIF5C proteins

• Reduced antibody performance over time:

  • Solution: Avoid repeated freeze-thaw cycles

  • Store according to manufacturer recommendations (typically at -20°C with 0.02% sodium azide and 50% glycerol)

• Inconsistent results across different sample types:

  • Solution: Optimize extraction and detection protocols for each sample type

  • Verify KIF5B expression levels in your specific samples before detailed studies

• Interference from post-translational modifications:

  • Solution: Be aware that KIF5B undergoes modifications such as arginine methylation

  • Use modification-specific antibodies when studying these aspects

• Non-specific nuclear staining:

  • Solution: Optimize blocking conditions and antibody dilutions

  • Include appropriate controls to distinguish specific from non-specific signals

How do I differentiate between endogenous KIF5B and overexpressed constructs in my experiments?

When working with both endogenous KIF5B and overexpressed constructs:

• Tagged KIF5B constructs: Use epitope tags (FLAG, GFP, etc.) that can be detected with tag-specific antibodies:

  • Example: FLAG-tagged KIF5B can be detected with anti-FLAG antibodies

  • GFP-tagged KIF5B can be visualized directly by fluorescence microscopy

• Molecular weight differences: Tagged KIF5B constructs will have slightly higher molecular weights than endogenous KIF5B:

  • Endogenous KIF5B: 110-120 kDa

  • GFP-tagged KIF5B: ~137 kDa (GFP adds ~27 kDa)

  • FLAG-tagged KIF5B: ~111-112 kDa (FLAG tag adds ~1-2 kDa)

• Distinguish native from exogenous expression: In rescue experiments, use KIF5B knockout cells expressing tagged KIF5B to ensure all detected KIF5B is from the exogenous construct .

• Quantitative analysis: Use Western blotting to quantify the relative levels of endogenous versus overexpressed KIF5B to ensure physiologically relevant expression levels.

How can KIF5B antibodies be used to investigate its role in disease models?

KIF5B has been implicated in several disease contexts:

• Cancer research: KIF5B gene rearrangements involving fusions to ALK and RET have been identified as drivers for lung cancer and other malignancies :

  • Use antibodies specific to KIF5B-ALK fusion proteins

  • Investigate expression patterns in tumor versus normal tissues

• Neurological disorders: Given KIF5B's role in neurons, investigate:

  • Expression levels in neurodegenerative disease models

  • Localization changes in pathological conditions

  • Altered interactions with cargo proteins in disease states

• Cardiovascular research: KIF5B is involved in trafficking of the Kv1.5 cardiac potassium channel:

  • Study the impact of KIF5B dysfunction on cardiac ion channel distribution

  • Investigate potential links to arrhythmias or other cardiac pathologies

What advanced imaging techniques can be combined with KIF5B antibodies to study its dynamics?

Several advanced imaging approaches can enhance KIF5B research:

• Live cell imaging: Combine fixed-cell antibody staining with live imaging of fluorescently tagged KIF5B to correlate dynamic behavior with endogenous protein distribution .

• Super-resolution microscopy: Techniques such as STORM, PALM, or STED can provide nanoscale resolution of KIF5B localization relative to microtubules and cargo.

• FRAP (Fluorescence Recovery After Photobleaching): Study the mobility and turnover of KIF5B at specific cellular locations.

• Single-molecule tracking: Investigate the movement of individual KIF5B molecules along microtubules.

• Correlative light and electron microscopy (CLEM): Combine antibody-based fluorescence imaging with ultrastructural analysis to study KIF5B in the context of cellular ultrastructure.