KIF5B Antibody, FITC conjugated

What is KIF5B and what are its primary cellular functions?

KIF5B (kinesin family member 5B), also known as conventional kinesin heavy chain or ubiquitous kinesin heavy chain (UKHC), is a microtubule-dependent motor protein with a calculated molecular weight of 110 kDa (observed at 110-120 kDa on Western blots). It consists of three structural domains: a globular N-terminal motor domain, a central alpha-helical rod domain, and a globular C-terminal domain .

KIF5B plays crucial roles in multiple cellular processes including:

• Transport of mitochondria and lysosomes to maintain their normal distribution

• Regulation of centrosome and nuclear positioning during mitotic entry

• Antagonism of dynein function during G2 phase to drive nuclei and centrosome separation

• Anterograde axonal transportation of proteins like MAPK8IP3/JIP3 for axon elongation

• Direction of lysosome movement toward microtubule plus ends through interaction with PLEKHM2 and ARL8B

• Facilitation of NK cell-mediated cytotoxicity by driving polarization of cytolytic granules

• Dendritic transport of RNA-binding proteins like FMRP, which is essential for synaptic plasticity and memory formation

What applications is a FITC-conjugated KIF5B antibody most suitable for?

A FITC-conjugated KIF5B antibody is particularly valuable for fluorescence-based applications where direct visualization of KIF5B is required. The most suitable applications include:

• Immunofluorescence microscopy/Immunocytochemistry (IF/ICC): Recommended dilution ranges from 1:50-1:800 depending on the specific antibody formulation

• Flow cytometry (intracellular): For quantitative analysis of KIF5B expression in cell populations

• Live cell imaging: For tracking KIF5B-mediated transport dynamics in real-time

• Fluorescence co-localization studies: To examine KIF5B interactions with potential cargo proteins

The FITC conjugation eliminates the need for secondary antibody incubation, reducing experiment time and potential background signal issues in multi-labeling experiments.

What are the optimal storage conditions for maintaining FITC-conjugated KIF5B antibody activity?

To maintain optimal activity of FITC-conjugated KIF5B antibodies:

• Store at -20°C in darkness (FITC is light-sensitive)

• Use storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Most formulations remain stable for one year after shipment when properly stored

• For small volume antibodies (e.g., 20µl sizes), aliquoting is unnecessary for -20°C storage

• Some formulations may contain 0.1% BSA as a stabilizer

When working with the antibody:

• Avoid repeated freeze-thaw cycles

• Protect from prolonged light exposure to prevent photobleaching of the FITC conjugate

• Allow to equilibrate to room temperature before opening the vial

How can I distinguish between KIF5B and other kinesin family members in microscopy studies?

Distinguishing between KIF5B and other kinesin family members (particularly KIF5A and KIF5C) is critical for accurate interpretation of experimental results:

• Antibody selection is crucial: Choose antibodies raised against the diverse carboxyl-terminal regions of KIF5 proteins. The tail domains of KIF5A, KIF5B, and KIF5C have distinct sequences that determine their functional specificity .

• Validation through knockout/knockdown controls: Include KIF5B-specific knockdown or knockout samples as controls. Studies have shown that KIF5B depletion affects specific cargoes like FMRP, while KIF5A knockdown has different effects .

• Expression pattern analysis: KIF5B is ubiquitously expressed, while KIF5A and KIF5C show more restricted expression patterns, particularly in neurons. This can help differentiate the proteins in certain tissue contexts.

• Cargo co-localization: KIF5B specifically interacts with certain cargoes. For example, pull-down experiments show that FMRP is preferentially pulled down by KIF5B and KIF5C, but not KIF5A .

For quantitative distinction, use high-resolution microscopy techniques with careful colocalization analysis and appropriate controls.

What are the methodological considerations for studying KIF5B-mediated transport of FMRP?

Studying KIF5B-mediated transport of FMRP requires careful experimental design:

• Live imaging approach:

  • Transfect neurons with GFP-FMRP along with control or KIF5B-specific shRNA

  • Conduct live imaging at one frame per second for 100 seconds

  • Analyze movement by creating kymographs to classify different movement types (stationary, oscillatory, bidirectional, anterograde, retrograde)

• Movement quantification:

  • Categorize FMRP granule movements into distinct classes

  • Compare densities of granules showing each category of movement between control and KIF5B-depleted conditions

• Specificity controls:

  • Include KIF5A knockdown as a comparison to demonstrate specificity

  • Pull-down assays with GST-tagged KIF5s can confirm direct interaction between KIF5B and FMRP

• Functional validation:

  • Assess synaptic plasticity and memory formation in conditional KIF5B knockout mice

  • Examine dendritic spine structural plasticity in vivo to connect molecular transport defects to cognitive outcomes

This approach reveals that KIF5B specifically regulates FMRP transport, and its depletion leads to deficits in dendritic transport, synaptic plasticity, and memory formation.

How can I optimize dual-color imaging involving FITC-conjugated KIF5B antibody and other fluorophores?

Optimizing dual-color imaging with FITC-conjugated KIF5B antibody requires addressing several technical considerations:

• Fluorophore selection to minimize spectral overlap:

  Second Fluorophore	Excitation (nm)	Emission (nm)	Compatibility with FITC
  DAPI	358	461	Excellent
  TRITC/Cy3	550	570	Good
  Cy5/Alexa 647	650	670	Excellent
  mCherry	587	610	Good

• Sequential acquisition to minimize bleed-through between channels, especially when using fluorophores with closer spectral properties

• Sample preparation optimizations:

  • For tissue sections: Perform antigen retrieval with TE buffer (pH 9.0) or alternatively with citrate buffer (pH 6.0)

  • For cell cultures: Optimize fixation (4% PFA for 15 minutes at room temperature generally preserves KIF5B localization well)

• Controls for spectral unmixing:

  • Include single-stained controls for each fluorophore

  • Prepare an unstained sample for autofluorescence correction

• Image processing considerations:

  • Apply appropriate channel alignment corrections

  • Use deconvolution algorithms if available to improve signal-to-noise ratio

  • Employ colocalization analysis tools with appropriate statistical measures (e.g., Pearson's correlation coefficient)

What are the recommended protocols for detecting KIF5B in different sample types?

Protocols should be optimized based on sample type and application:

For Western Blotting:

• Recommended dilution: 1:2000-1:50000 (depending on antibody formulation)

• Successfully detected in: A549 cells, HeLa cells, HepG2 cells, Jurkat cells, mouse brain, mouse heart, rat brain

• Sample preparation: Lyse cells in RIPA buffer with protease inhibitors, denature at 95°C for 5 minutes in loading buffer

• Loading: 20-50 μg total protein per lane depending on expression level

For Immunohistochemistry:

• Recommended dilution: 1:20-1:2000 (antibody-dependent)

• Antigen retrieval: TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0

• Successfully detected in: human kidney tissue, human skin tissue, human ovarian cancer tissues

• Blocking: 10% normal serum in PBS for 1 hour at room temperature

• Primary antibody incubation: Overnight at 4°C

For Immunofluorescence/ICC:

• Recommended dilution: 1:50-1:800

• Successfully detected in: HepG2 cells

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.1% Triton X-100 for 10 minutes

• Blocking: 5% BSA in PBS for 1 hour

• Primary antibody incubation: Overnight at 4°C or 2 hours at room temperature

How can I quantitatively analyze KIF5B-mediated transport in live neurons?

Quantitative analysis of KIF5B-mediated transport requires specialized approaches:

• Live imaging setup:

  • Transfect neurons with fluorescently-tagged cargo proteins (e.g., GFP-FMRP)

  • Use a temperature-controlled chamber (37°C, 5% CO2)

  • Acquire time-lapse images at 1 frame/second for 100 seconds

  • Focus on dendritic segments 50-100 μm from the cell body

• Movement categorization system:

  Movement Type	Definition	Velocity Range
  Stationary	<0.1 μm/min displacement	-
  Oscillatory	Bidirectional with <2 μm net movement	-
  Anterograde	>2 μm movement toward distal dendrites	0.2-2.5 μm/s
  Retrograde	>2 μm movement toward soma	0.2-2.5 μm/s
  Bidirectional	Combination of significant anterograde and retrograde movements	Variable

• Quantification methods:

  • Generate kymographs for visual representation of movement

  • Calculate the following parameters:

    • Granule density (granules/100 μm)

    • Percentage of mobile vs. immobile granules

    • Velocity distributions

    • Run lengths

    • Directional bias (anterograde vs. retrograde)

• Perturbation approaches:

  • KIF5B knockdown via shRNA

  • Dominant negative constructs

  • Conditional knockout in specific neuronal populations

This approach has successfully demonstrated the specific role of KIF5B in FMRP granule transport and its functional impact on synaptic plasticity.

What are the key considerations for IP experiments targeting KIF5B and its cargo interactions?

Successful immunoprecipitation (IP) of KIF5B and identification of its cargo interactions requires:

• Sample preparation:

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

  • Prepare lysates in non-denaturing buffers that preserve protein-protein interactions (e.g., NP-40 buffer with protease inhibitors)

  • Include phosphatase inhibitors if studying phosphorylation-dependent interactions

  • Perform pre-clearing step with protein A/G beads to reduce non-specific binding

• Antibody selection:

  • Choose antibodies validated for IP applications

  • Consider using different antibodies for IP and detection to avoid heavy/light chain interference

  • For FITC-conjugated antibodies in IP, be aware that the conjugation may affect antigen recognition

• Controls to include:

  • IgG control (same species as KIF5B antibody)

  • Input sample (5-10% of lysate used for IP)

  • For RNA-protein interactions (e.g., KIF5B-FMRP), include RNase-treated samples

• Analysis of cargo interactions:

  • Western blot for known/suspected interaction partners

  • Mass spectrometry for unbiased identification of novel interactors

  • RNA sequencing for identifying transported mRNAs in KIF5B-RNP complexes

• Validation approaches:

  • Reverse IP (immunoprecipitate cargo and probe for KIF5B)

  • GST pull-down assays with recombinant proteins

  • Proximity ligation assays to confirm interactions in situ

This approach has been successfully used to demonstrate KIF5B's preferential interaction with FMRP compared to KIF5A, highlighting the functional specificity of different kinesin family members .

Why might I observe high background when using FITC-conjugated KIF5B antibody in immunofluorescence?

High background with FITC-conjugated KIF5B antibodies can result from several factors:

• Antibody concentration issues:

  • Too high concentration of primary antibody (start with 1:200-1:800 dilution and optimize)

  • Inadequate washing between steps (use at least 3x5-minute washes with PBS-T)

• Fixation and permeabilization problems:

  • Overfixation can increase autofluorescence

  • Excessive permeabilization may expose non-specific binding sites

  • Recommended: 4% PFA for 15 minutes followed by 0.1% Triton X-100 for 10 minutes

• Blocking insufficiency:

  • Insufficient blocking (use 5% BSA or 10% normal serum from the same species as secondary antibody)

  • Inadequate blocking time (minimum 1 hour at room temperature)

• FITC-specific issues:

  • Photobleaching (minimize exposure to light during all steps)

  • Autofluorescence (particularly in fixed tissues - consider using longer wavelength fluorophores)

  • pH sensitivity (FITC fluorescence is optimal at pH 8.0-9.0)

• Sample-specific considerations:

  • Lipofuscin autofluorescence in aging tissues (use Sudan Black B treatment)

  • Aldehyde-induced autofluorescence (treat with sodium borohydride)

  • Endogenous biotin/avidin binding (block with avidin/biotin blocking kit if using biotin-based detection)

For reliable results with minimal background, titrate the antibody concentration for each application and include appropriate negative controls (no primary antibody and isotype control).

How can I validate the specificity of my KIF5B antibody signal?

Validating KIF5B antibody specificity requires multiple complementary approaches:

• Genetic controls:

  • KIF5B knockdown using validated shRNAs

  • KIF5B knockout cells/tissues (conditional KO models are available)

  • Compare against related kinesins (KIF5A, KIF5C) to confirm specificity

• Biochemical validation:

  • Western blot should show a single band at 110-120 kDa

  • Pre-absorption with immunizing peptide should abolish signal

  • Mass spectrometry confirmation of immunoprecipitated protein

• Application-specific controls:

  • For IF/ICC: Co-staining with multiple KIF5B antibodies targeting different epitopes

  • For WB: Positive controls from validated expressive tissues (brain, heart)

• Cross-species validation:

  • Antibodies have shown reactivity with human, mouse, and rat samples

  • Some have reported reactivity with pig and monkey samples as well

• Function-based validation:

  • Confirm expected subcellular localization

  • Verify expected alterations in localization or expression under perturbation conditions

  • Correlate with functional phenotypes (e.g., FMRP transport, mitochondrial distribution)

For FITC-conjugated antibodies specifically, compare signals with unconjugated primary plus FITC-conjugated secondary antibody detection to ensure conjugation hasn't affected specificity.

What are the common pitfalls when studying KIF5B in neuronal transport systems?

Studying KIF5B in neuronal transport involves several potential pitfalls:

• Functional redundancy misinterpretation:

  • Despite high sequence homology, KIF5 family members (KIF5A, KIF5B, KIF5C) have non-redundant functions

  • KIF5B specifically affects FMRP transport, while KIF5A knockdown shows different effects

  • Always include specific controls for each family member to avoid misattribution of function

• Static vs. dynamic analysis limitations:

  • Fixed sample imaging fails to capture transport dynamics

  • Live imaging requires careful optimization of imaging frequency and duration

  • Balance between temporal resolution and photobleaching/phototoxicity

• Overexpression artifacts:

  • Overexpressed motor proteins may form non-physiological aggregates

  • Can sequester endogenous binding partners

  • May saturate regulatory mechanisms

  • Consider using genome editing for tagging endogenous proteins

• Developmental timing considerations:

  • KIF5B function changes during neuronal maturation

  • Adult conditional knockout mice show memory and synaptic plasticity deficits

  • Embryonic lethality of complete knockout limits some analyses

• Technical imaging challenges:

  • Kinesin transport occurs in three dimensions

  • Standard 2D imaging may miss vertical movements

  • Use z-stacks or specialized 3D tracking techniques for complete analysis

• Cargo complexity:

  • KIF5B transports multiple cargoes (mitochondria, lysosomes, mRNPs)

  • Effects on one cargo type may be secondary to effects on another

  • Use cargo-specific markers and co-transport analysis

To overcome these challenges, employ complementary approaches including conditional genetic models, rescue experiments, and correlative structural-functional analyses.

How does post-translational modification regulate KIF5B function?

Post-translational modifications (PTMs) are crucial regulators of KIF5B function:

• Arginine methylation:

  • Recently identified as a novel PTM for KIF5B

  • Regulates cargo binding specificity

  • May explain differential cargo preferences between KIF5 family members

• Phosphorylation:

  • Multiple phosphorylation sites have been identified

  • Affects motor activity and cargo binding

  • JNK pathway may regulate KIF5B through phosphorylation

  • Can be studied using phospho-specific antibodies and phosphomimetic mutations

• Ubiquitination:

  • Regulates KIF5B degradation and turnover

  • May control local availability of transport machinery

  • Proteasome inhibitors can be used to assess ubiquitination-dependent regulation

• Other modifications:

  • Acetylation may affect microtubule binding

  • SUMOylation has been reported for some kinesins

  • Oxidative modifications can occur under stress conditions

To study PTMs of KIF5B:

• Use PTM-specific antibodies

• Employ mass spectrometry for unbiased PTM mapping

• Generate mutants that mimic or prevent specific modifications

• Apply specific enzyme inhibitors to alter PTM status

Understanding these modifications provides insight into the regulation of KIF5B's motor activity, cargo selectivity, and functional specificity in different cellular contexts.

What is the role of KIF5B in neurodegenerative disease models?

KIF5B dysfunction has been implicated in several neurodegenerative conditions:

• Alzheimer's Disease:

  • KIF5B-mediated transport of APP and BACE1 affects amyloid production

  • Disrupted axonal transport precedes other pathological features

  • Potential therapeutic target for early intervention

• Fragile X Syndrome:

  • KIF5B specifically transports FMRP-containing granules

  • KIF5B conditional knockout mice show deficits in synaptic plasticity and memory

  • Similar phenotypes to FMRP-deficient models

  • Suggests shared pathological mechanisms

• ALS and motor neuron diseases:

  • Long motor neurons particularly vulnerable to transport defects

  • KIF5B mediates mitochondrial transport, critical for axonal health

  • Mutations in transport machinery components cause motor neuron degeneration

• Parkinson's Disease:

  • KIF5B involved in transport of mitochondria and lysosomes

  • Both organelles critical for maintaining dopaminergic neuron health

  • Potential link to PINK1/Parkin pathway through mitochondrial dynamics

Research approaches:

• Analyze KIF5B expression and localization in disease tissues

• Assess transport dynamics in patient-derived neurons

• Test whether restoring KIF5B function rescues disease phenotypes

• Develop transport-targeted therapeutic approaches

This emerging research suggests KIF5B as both a contributor to pathogenesis and potential therapeutic target in multiple neurodegenerative conditions.

How do advanced imaging techniques enhance our understanding of KIF5B function?

Advanced imaging techniques have revolutionized KIF5B research:

• Super-resolution microscopy:

  • STED, STORM, and PALM overcome diffraction limit

  • Resolve KIF5B-cargo complexes at nanometer resolution

  • Can visualize individual motor proteins on microtubules

  • Reveal nanoscale organization of transport machinery

• Single-molecule tracking:

  • Directly observe individual KIF5B motors in living cells

  • Measure parameters including:

    Parameter	Typical Value	Method
    Velocity	0.2-2.5 μm/s	Particle tracking
    Run length	0.5-5 μm	Single-molecule kymography
    Force generation	5-7 pN	Optical trapping
    Step size	8 nm	High-precision tracking

• FRET-based sensors:

  • Monitor KIF5B conformational changes during transport cycle

  • Detect cargo binding/release events

  • Measure regulatory modifications in real-time

• Correlative light-electron microscopy (CLEM):

  • Connect fluorescence observations with ultrastructural context

  • Visualize KIF5B in relation to organelles and cytoskeletal elements

  • Bridge functional observations with structural details

• Light-sheet microscopy:

  • Reduced phototoxicity for long-term imaging

  • Visualize transport dynamics throughout entire neurons

  • Track multiple cargoes simultaneously in 3D

• Expansion microscopy:

  • Physical expansion of specimens for improved resolution

  • Compatible with standard microscopes

  • Reveals details of KIF5B-cargo complexes

These advanced techniques have revealed that KIF5B functions not as an isolated motor but within complex macromolecular assemblies whose composition and organization dynamically change to regulate transport in different cellular contexts and in response to various stimuli.