KIF17 Antibody, Biotin conjugated

What is KIF17 and why is it important in neuroscience research?

KIF17 is a kinesin superfamily member encoding a neuron-specific molecular motor. In neurons, KIF17 plays a critical role in intracellular transport, specifically the specialized transport of:

• N-methyl-D-aspartate receptor NR2B subunit

• Glutamate receptor GluR5

• Potassium channel Kv4.2

KIF17 transports these cargo molecules from the cell body to dendrites . This transport function is particularly significant for synaptic plasticity and neuronal morphogenesis. KIF17 has been implicated in learning and memory processes through its role in regulating NMDA receptor trafficking .

Recent research has also identified KIF17's involvement in:

• Photoreceptor outer segment turnover

• Microtubule stabilization in epithelial cells

• Cerebellar development via Sonic Hedgehog signaling pathways

What applications is the biotin-conjugated KIF17 antibody suitable for?

The biotin-conjugated KIF17 antibody is primarily optimized for ELISA (Enzyme-Linked Immunosorbent Assay) applications . Unlike its unconjugated counterparts which can be used in Western Blot, Immunohistochemistry, and Immunofluorescence, the biotin-conjugated version is more specialized.

The biotin-conjugated format offers enhanced detection sensitivity in ELISA applications due to the strong binding affinity between biotin and streptavidin .

What are the recommended storage conditions for KIF17 antibody, biotin conjugated?

For optimal performance and longevity of the biotin-conjugated KIF17 antibody, follow these research-validated storage protocols:

• Store at -20°C for long-term storage

• Aliquot upon receipt to avoid repeated freeze/thaw cycles

• Avoid exposure to light as this can degrade the biotin conjugate

• The antibody remains stable for approximately 12 months from date of receipt when properly stored

• Some preparations may contain 50% glycerol as a cryoprotectant with 0.03% Proclin-300 as preservative

For working solutions:

• Keep at 2-8°C for up to two weeks during active experimental periods

• Discard working dilutions if not used within 12 hours

How does phosphorylation of KIF17 regulate its function and localization in neurons?

Phosphorylation of KIF17 represents a critical regulatory mechanism that significantly impacts its subcellular localization and cargo transport functions. Research has identified a conserved phosphorylation site (S1029 in mice and S815 in zebrafish) adjacent to a nuclear localization signal (NLS) in the C-terminus .

Key research findings on KIF17 phosphorylation:

• Enhanced ciliary/outer segment localization: Phospho-mimetic KIF17 (S1029D in mouse or S815D in zebrafish) shows significantly increased localization to neuronal cilia and photoreceptor outer segments compared to wild-type or phospho-deficient (S1029A/S815A) KIF17 .

• CaMKII-mediated regulation: Ca²⁺/calmodulin-dependent protein kinase phosphorylates the tail domain of KIF17 at S1029, attenuating its interaction with cargo adapter proteins like Mint1 .

• Impact on cargo release: Phosphorylation at S1029 reduces the interaction between KIF17 and Mint1, potentially facilitating the release of cargo vesicles containing NMDA receptor subunits at their destination .

• Physiological significance: In photoreceptors, expression of phospho-mimetic KIF17(S815D) leads to a two-fold increase in cone disc shedding, while genetic mutants of KIF17 in zebrafish and mice show diminished disc shedding .

• Ciliary vs. nuclear localization: Phosphorylation appears to promote ciliary localization while potentially inhibiting nuclear localization in a cell type-dependent manner .

Experimental evidence from transgenic animals demonstrates that phosphorylation state-specific mutations in KIF17 (S1029A/D or S815A/D) have significant phenotypic consequences for neuronal communication and photoreceptor maintenance .

What are the optimal biotinylation approaches for enhancing KIF17 antibody sensitivity in ELISA assays?

Optimization of biotinylation strategy is crucial for maximizing sensitivity in ELISA assays using KIF17 antibodies. Research comparing different biotinylation reagents and protocols has revealed significant variations in assay performance:

• Impact of biotinylation reagents: Different biotinylation chemistry can affect antibody performance by up to an order of magnitude in sensitivity . When selecting a biotinylation approach for KIF17 antibody, consider:

  Biotinylation Reagent Type	Advantages	Considerations
  NHS-ester biotinylation reagents	Common, versatile conjugation to primary amines	Can modify lysines in antigen-binding sites
  Maleimide-activated biotin reagents	Site-specific conjugation to thiols	Requires reduced antibody preparation
  Photoactivatable biotin reagents	Less disruptive to binding sites	Requires UV exposure, potentially damaging

• Molar fold excess optimization: The ratio of biotin reagent to antibody (molar fold excess) significantly impacts performance . For KIF17 antibodies:

  • Too low: Insufficient labeling resulting in reduced sensitivity

  • Too high: Over-labeling can interfere with antigen binding

  • Optimal range: Typically 5-20 fold molar excess, but requires empirical determination for each antibody preparation

• Verification of biotinylation efficiency: Critical step for ensuring consistent assay performance . Methods include:

  • HABA assay for biotin quantification

  • ELISA-based biotinylation quantification kits

  • Lateral flow biotinylation detection kits

• Purification post-biotinylation: Removal of excess unreacted biotin is essential to prevent interference with streptavidin binding:

  • Dialysis against PBS (at least 1000× volume, multiple changes)

  • Size-exclusion chromatography

  • Protein G purification can help maintain antibody activity post-biotinylation

Research findings indicate that optimized biotinylation can enhance detection sensitivity by 5-20 fold compared to suboptimal approaches for antibodies in general , making this a critical consideration for KIF17 antibody-based ELISA development.

How can immunoprecipitation protocols be optimized when using KIF17 antibody for studying protein-protein interactions?

Optimization of immunoprecipitation (IP) protocols is essential when studying KIF17 interactions with binding partners such as Mint1, NR2B, GluR5/6, and other adaptor proteins. The following methodological considerations are based on published research:

• Tissue/cell preparation: For KIF17 IP from brain tissue, specialized preparation is critical:

  • Homogenize tissue in RIPA buffer containing phosphatase and protease inhibitors

  • Centrifuge at 13,000 × g for 20 min at 4°C

  • Determine protein concentration by BCA protein assay

• Key co-immunoprecipitation findings:

  • KIF17 interacts with GluR6/7 and KA2 subunits in hippocampal preparations

  • KIF17 binds to APC through its stalk domain independent of EB1

  • Phosphorylation at S1029 attenuates the interaction between KIF17 and Mint1

• Detection considerations:

  • KIF17 typically appears at 170 kDa on immunoblots despite a calculated molecular weight of 115 kDa

  • Use densitometry analysis with ImageJ software to quantify co-IP results

• Controls:

  • Include knockouts when available (e.g., GluR5−/−, KA2−/−, GluR6/7−/−) to verify specificity

  • Include negative controls such as IgG and non-related proteins (KIF3, GluR2) to demonstrate specificity

What methodological approaches can address the challenges of studying KIF17 in photoreceptor cells using biotin-conjugated antibodies?

Studying KIF17 in photoreceptor cells presents unique challenges due to the specialized structure of these cells and dynamic processes like disc shedding. When using biotin-conjugated KIF17 antibodies in photoreceptor research, consider these methodological approaches:

• Tissue preparation and fixation:

  • For immunohistochemistry, suggested antigen retrieval with TE buffer pH 9.0 has been validated for KIF17 detection in retinal tissue

  • Alternative approach: antigen retrieval with citrate buffer pH 6.0

  • For zebrafish retina, consider 4% paraformaldehyde fixation for 2 hours at room temperature

• Enhanced visualization strategies:

  • Use tyramide signal amplification (TSA) to enhance signal from biotinylated antibodies

  • Consider dual labeling with established photoreceptor markers:

    • Rhodopsin for rod outer segments

    • Cone transducin-α for cone outer segments

• Controls for phosphorylation studies:

  • Compare wild-type KIF17-GFP with phospho-mimetic (S815D) and phospho-deficient (S815A) transgenes

  • Include genetic mutants of KIF17 (kif17-/- zebrafish or mice)

  • Expression of constitutively active tCaMKII can serve as a positive control for KIF17 phosphorylation

• Imaging considerations:

  • For detailed localization within photoreceptor outer segments, high-resolution confocal microscopy with deconvolution is recommended

  • Z-stack imaging through the entire outer segment is crucial to distinguish proximal versus distal localization patterns

How can researchers optimize biotinylated KIF17 antibody use in studying neuronal cargo transport mechanisms?

Investigating KIF17's role in neuronal cargo transport requires specialized methodological approaches. When using biotinylated KIF17 antibodies in these studies, consider these research-validated optimization strategies:

• Visualizing KIF17-cargo complexes:
  Research has identified specific KIF17-cargo associations that can be studied using optimized protocols:

  Cargo	Adaptor Proteins	Validated Detection Methods
  NMDA receptor NR2B	Mint1/CASK/Velis	Co-IP, IF colocalization
  Glutamate receptor GluR5	Unknown	Co-IP, transport assays
  Kv4.2 potassium channel	Unknown	Co-IP, transport assays

• Live imaging optimization:

  • For live-cell visualization of KIF17-mediated transport, consider:

    • Photoconversion approaches with mEOS-tagged KIF17

    • FRAP (Fluorescence Recovery After Photobleaching) to measure transport dynamics

    • Dual-color imaging with cargo labeled with complementary fluorophores

  • When using biotin-conjugated antibodies, pre-complexing with fluorescent streptavidin before application to permeabilized cells can enable specific visualization

• Verifying specificity of transport:

  • Use dominant-negative KIF17 lacking its motor domain as a control

  • Compare with other kinesin family members (KIF3) to demonstrate cargo specificity

  • Utilize genetic knockouts (GluR6−/−, KA2−/−) to verify components of transport complexes

• Detection optimization for biotin-conjugated antibodies:

  • For post-fixation detection, the streptavidin-HRP system provides enhanced sensitivity

  • For immunofluorescence applications requiring signal amplification, consider:

    • Using streptavidin-conjugated quantum dots for photostable signal

    • Tyramide signal amplification for detection of low-abundance complexes

    • Multiple layers of amplification (biotin-streptavidin-biotin) for ultra-sensitive detection

What are the most effective methods to verify biotinylation efficiency of KIF17 antibodies?

Verifying biotinylation efficiency is critical for ensuring consistent experimental results with biotin-conjugated KIF17 antibodies. Several validated methodologies can be employed:

• Quantitative biotin determination:

  • HABA/Avidin-based spectrophotometric assays

  • Mass spectrometry analysis for precise determination of biotin:antibody ratio

• Functional verification:

  • Competitive binding assay with known biotinylated standards

  • Standard curve comparison between different batches of biotinylated antibody

  • Side-by-side comparison with unconjugated antibody in non-biotin-dependent applications

• Quality control criteria:

  Parameter	Acceptable Range	Method of Determination
  Biotin:Antibody ratio	3-8 biotin molecules per antibody	HABA assay
  Retained immunoreactivity	>80% of unconjugated antibody	Comparative ELISA
  Free biotin contamination	<5% of total biotin	Dialysis assessment
  Antibody recovery post-labeling	>85% of starting material	Protein concentration determination

• Optimizing biotinylation reaction:
  Research has shown that varying the molar fold excess biotin during the biotinylation process can highly affect the performance of immunoassays, with differences of up to an order of magnitude in sensitivity .

How can researchers troubleshoot inconsistent results when using KIF17 antibody, biotin conjugated in ELISA?

When encountering inconsistent results with biotin-conjugated KIF17 antibody in ELISA, systematic troubleshooting should address these key methodological factors:

• Antibody-specific issues:

• Protocol optimization:

• Technical considerations:

• Validation approach:

  Problem	Diagnostic Test	Solution
  Low signal	Standard curve with recombinant KIF17	Increase antibody concentration; enhance detection system
  High background	No-primary control wells	Optimize blocking; increase washing stringency
  Poor reproducibility	Calculate %CV between replicates (target <15%)	Standardize pipetting; use calibrated equipment
  Hook effect	Serial dilution of high-concentration samples	Include multiple sample dilutions in analysis

What are the critical considerations when interpreting results from KIF17 antibody experiments in neurodevelopmental studies?

Interpreting results from KIF17 antibody experiments in neurodevelopmental studies requires careful consideration of several biological and technical factors:

• Developmental timing effects:

  • KIF17 expression increases over developmental time, as observed in cerebellar studies

  • Consider including multiple developmental timepoints (e.g., P7, P10, P21) when studying KIF17 in developing neural tissues

  • Kif17 germline deletions may have different phenotypic consequences at different developmental stages

• Cell-type specificity:

  • KIF17 functions differently in different neuronal populations:

    • In Purkinje cells: Promotes Sonic Hedgehog signaling

    • In cerebellar granule neuron precursors: Restricts Hedgehog signaling through regulation of GLI transcription factors

  • Verify cell-type specific expression using co-localization with established markers

• Phosphorylation state considerations:

  • KIF17 phosphorylation at S1029/S815 affects localization and function

  • Consider using phospho-specific antibodies or phospho-mimetic/deficient constructs

  • Activation of CaMKII can lead to KIF17 phosphorylation and altered function

• Genetic background effects:

  • Consider potential compensatory mechanisms in knockout models

• Technical validation requirements:

  • Verify antibody specificity using KIF17 knockout tissues as negative controls

  • Include multiple antibodies targeting different epitopes when possible

  • For biotin-conjugated antibodies, include controls for non-specific streptavidin binding

  • Consider potential detection of KIF17 splice variants (observed molecular weight: 170 kDa vs. calculated: 115 kDa)

How can KIF17 antibodies be utilized in studying microtubule dynamics and stabilization?

Recent research has revealed that KIF17 plays unexpected roles in microtubule stabilization, suggesting novel applications for KIF17 antibodies in studying cytoskeletal dynamics:

• KIF17's role in microtubule stability:

  • KIF17 localizes to microtubule plus ends and contributes to microtubule stabilization

  • KIF17 knockdown reduces acetylated tubulin levels (a marker of stabilized microtubules) by approximately 57%

  • Overexpression of active KIF17 increases acetylated microtubules

• Protein interaction studies:

  • KIF17 interacts with EB1 and APC at microtubule plus ends

  • Biotin-conjugated KIF17 antibodies can be used in pull-down assays to identify interaction partners

  • Research has shown that KIF17 binds APC through its stalk domain independently of EB1

  KIF17 Domain	Interaction Partners	Functional Significance
  Motor domain	Microtubules	Movement along microtubules
  Stalk domain	APC	Microtubule stabilization
  Tail domain	Mint1, cargo adaptors	Cargo binding and release

• Methodological considerations:

  • Use biotin-conjugated KIF17 antibodies in combination with proximity ligation assays to visualize interactions with EB1 and APC

  • For live imaging of KIF17 at microtubule plus ends, consider:

    • Photoconvertible tags for pulse-chase experiments

    • FRAP for measuring turnover rates at plus ends

    • Dual-color imaging with EB1/APC

• Relevant mutants for mechanistic studies:

  • KIF17 G754E: Rigor mutant that binds but doesn't move on microtubules

  • KIF17 G754E-G243A: Rigor motor that doesn't bind nucleotides

  • KIF17 G754E-R288/294A: Mutant that doesn't bind microtubules

How do different formulations of biotin-conjugated KIF17 antibodies compare in specialized neuroscience applications?

Different formulations of biotin-conjugated KIF17 antibodies may exhibit varying performance characteristics in specialized neuroscience applications. The following methodological comparison is based on research findings:

• Buffer formulation effects:
  Common buffer formulations for biotin-conjugated KIF17 antibodies include:

  Buffer Components	Advantages	Limitations	Applications
  PBS with 0.02% sodium azide and 50% glycerol, pH 7.3	Long-term stability; prevents freeze-thaw damage	Higher viscosity; dilution required	Long-term storage; applications requiring concentrated antibody
  0.01 M PBS, pH 7.4, 0.03% Proclin-300 and 50% glycerol	Azide-free (compatible with HRP); extended shelf-life	Similar viscosity issues	Enzymatic assays sensitive to azide inhibition
  PBS without additives	Lower background in sensitive applications	Reduced stability; shorter shelf-life	Applications requiring minimal buffer components

• Conjugation chemistry comparison:
  The method of biotin attachment can significantly impact antibody performance:

  • NHS-ester biotinylation: Most common, targets primary amines

  • Maleimide-activated biotin: Targets reduced cysteines, potentially less disruptive to binding sites

  • Photoactivatable biotin: Allows spatiotemporal control of biotinylation

  Research has shown differences of up to an order of magnitude in sensitivity between different biotinylation reagents .

• Application-specific considerations:

  • For super-resolution microscopy: Consider smaller streptavidin conjugates (e.g., streptavidin-Alexa Fluor rather than streptavidin-HRP)

  • For electron microscopy: Streptavidin-gold conjugates offer excellent spatial resolution

  • For in vivo imaging: Consider streptavidin-NIR fluorophore conjugates for deeper tissue penetration

Research comparing different biotinylation approaches found that both the choice of biotinylation reagent and the molar fold excess biotin can highly affect the performance of assays, with sensitivity differences exceeding an order of magnitude .

What are the emerging applications of KIF17 antibodies in studying neurodevelopmental disorders?

KIF17's critical roles in neuronal transport and synaptic function position it as a relevant target for neurodevelopmental disorder research. Emerging applications for KIF17 antibodies in this field include:

• KIF17 in learning and memory disorders:
  KIF17-mediated transport of NMDA receptor NR2B subunits is implicated in learning and memory processes:

  • KIF17 phosphorylation at S1029 regulates interaction with Mint1 adapter protein

  • This modulates NR2B delivery to dendrites, affecting synaptic plasticity

  Research has shown that CaMKII phosphorylates KIF17, potentially linking calcium signaling, KIF17 function, and learning mechanisms .

• Retinal degeneration studies:
  KIF17 phosphorylation regulates photoreceptor outer segment turnover, positioning it as relevant for retinal degeneration disorders:

  • Phospho-mimetic KIF17 enhances disc shedding

  • KIF17 mutants show diminished disc shedding

  • Constitutively active CaMKII leads to KIF17-dependent increase in disc shedding

  This suggests potential therapeutic strategies targeting KIF17 phosphorylation in retinal degeneration disorders.

• Methodological considerations for neurodevelopmental research:

  • Cell-type specific analysis is critical due to KIF17's opposite functions in different neural populations

  • Developmental timing must be carefully controlled due to changing KIF17 expression patterns

  • Phosphorylation state-specific antibodies or constructs should be employed to distinguish active forms

  • Consider genetic interaction studies between KIF17 and established neurodevelopmental disorder genes

• Technical innovations for KIF17 detection in neurodevelopmental contexts:

  • Multiplexed immunofluorescence with cell-type markers and KIF17

  • Spatial transcriptomics correlated with KIF17 protein localization

  • Phospho-specific antibodies to distinguish active/inactive forms

  • Live imaging of KIF17-mediated transport in patient-derived neurons

Current research indicates that KIF17 expression increases over developmental time and that cerebellar weight is significantly reduced from P10 onward in KIF17 knockout models, highlighting critical developmental windows for KIF17 function .