KIF17 Antibody, FITC conjugated

What is KIF17 and what cellular functions does it regulate?

KIF17 is a kinesin family motor protein (kinesin family member 17) with a calculated molecular weight of 115 kDa, although it typically appears at approximately 170 kDa in experimental conditions . This homodimeric motor protein localizes at microtubule plus-ends where it contributes to microtubule stabilization and epithelial polarization . Recent research has revealed that KIF17 also localizes at cell-cell adhesions where it regulates RhoA-dependent actin remodeling . Additionally, KIF17 plays important roles in spermiogenesis, particularly in nuclear reshaping and sperm tail formation . The protein demonstrates autoinhibition via dual intramolecular mechanisms, with its C-terminal tail domain blocking microtubule binding in the absence of cargo .

What applications are appropriate for KIF17 antibodies in research?

KIF17 antibodies have been validated for multiple applications including Western Blot (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), Immunofluorescence (IF), and Co-Immunoprecipitation (CoIP) . The recommended dilutions for these applications vary:

When conducting experiments, it is crucial to titrate the antibody in each specific testing system to obtain optimal results, as reactivity may vary based on sample type and experimental conditions .

How do I optimize antigen retrieval for KIF17 detection in tissue sections?

For optimal KIF17 detection in tissue sections, antigen retrieval methodology significantly impacts staining quality. The recommended approach is to use TE buffer at pH 9.0 for antigen retrieval . This alkaline pH helps unmask epitopes by breaking protein cross-links formed during fixation. Alternatively, citrate buffer at pH 6.0 can be used, although this may result in different staining patterns or intensity . When optimizing your protocol, it's advisable to test both conditions in parallel with appropriate positive controls (such as brain tissue samples) to determine which works best for your specific tissue type and fixation conditions. Incubation time and temperature during antigen retrieval should also be optimized (typically 15-30 minutes at 95-100°C) to maximize signal while minimizing background and tissue damage.

How can I differentiate between specific and non-specific binding when using KIF17 antibodies in immunofluorescence studies?

Distinguishing specific from non-specific binding requires rigorous experimental controls and validation strategies. For KIF17 immunofluorescence studies, implement the following methodological approaches:

First, include a KIF17 knockout/knockdown control alongside your experimental samples. Published studies have utilized KIF17 knockdown systems that can serve as negative controls to identify non-specific binding patterns . Second, perform peptide competition assays where the KIF17 antibody is pre-incubated with excess KIF17 recombinant protein or immunizing peptide before application to samples; specific signals should be blocked while non-specific binding will remain. Third, compare staining patterns across multiple KIF17 antibodies targeting different epitopes - true KIF17 signals should show consistent localization patterns.

For FITC-conjugated antibodies specifically, include an isotype control antibody (same isotype, FITC-conjugated, non-targeting) to identify background fluorescence resulting from non-specific binding. Additionally, when examining KIF17 localization at cell-cell junctions or microtubule plus-ends, co-staining with established markers for these structures (E-cadherin for junctions, EB1 for plus-ends) can help validate specific localization patterns . Signal that fails to co-localize with known KIF17 interacting structures might represent non-specific binding.

What are the critical considerations when using KIF17 antibodies to study its dual roles in microtubule stabilization and actin remodeling?

Investigating KIF17's dual functionality requires specialized experimental design considerations. KIF17 localizes both at microtubule plus-ends and at cell-cell adhesions, where it influences distinct cytoskeletal elements . To effectively study these dual roles, consider the following methodological approaches:

For imaging studies, sequential or simultaneous co-labeling of microtubules (using α-tubulin antibodies) and actin (using phalloidin or actin antibodies) alongside KIF17 is essential. Note that phalloidin strongly labels stress fibers and bundled actin, which can mask non-bundled and branched filaments better detected with actin antibodies . When examining junctional actin specifically, consider implementing Sobel edge detection filters to enhance visualization of subtle actin enrichment at cell-cell contacts .

For functional studies, utilize truncation mutants of KIF17 to dissect domain-specific functions. The motor domain (e.g., K370 construct) affects junctional actin accumulation independently of microtubule binding, while tail domain truncations (e.g., 1-846 construct) affect microtubule binding properties . Expression of these constructs followed by live imaging with fluorescently tagged actin (GFP-actin or mCh-LifeAct) allows for real-time visualization of KIF17's effects on actin dynamics .

When manipulating RhoA signaling to study KIF17's actin-regulatory functions, use specific inhibitors of RhoA or ROCK, or expression of LIMK1 kinase-dead or activated cofilin S3A constructs, as these have been shown to inhibit KIF17-induced junctional actin accumulation .

How do I properly design co-immunoprecipitation experiments to identify novel KIF17 binding partners?

Designing effective co-immunoprecipitation (Co-IP) experiments for KIF17 requires careful consideration of antibody selection, buffer composition, and experimental controls. Based on published methodologies, follow these research-validated approaches:

First, select an antibody validated for IP applications – antibody 14615-1-AP has been successfully used for KIF17 immunoprecipitation at a concentration of 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate . For FITC-conjugated antibodies, confirm that conjugation doesn't interfere with the antibody's ability to recognize native protein conformations.

Regarding sample preparation, brain tissue has been successfully used for KIF17 IP experiments . Lysis buffers should maintain protein-protein interactions while effectively solubilizing membrane-associated proteins; a buffer containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), and protease inhibitors is recommended. For epithelial cell studies, consider that KIF17 interactions may be junction-specific, so avoid harsh detergents that might disrupt these associations .

To validate novel interactions, implement reciprocal Co-IPs where you immunoprecipitate with antibodies against the potential binding partner and blot for KIF17. Published studies have successfully used this approach to confirm protein interactions with KIF17 . For dimeric state analysis, co-express differentially tagged KIF17 constructs (e.g., Flag/Myc-KIF17 and fluorescent protein-tagged KIF17) and perform Co-IP with tag-specific antibodies .

Include appropriate controls: IgG control antibodies to identify non-specific binding , and competitive elution with immunizing peptides to confirm specificity of interactions.

What are the experimental considerations when using FITC-conjugated KIF17 antibodies for multi-color immunofluorescence microscopy?

When designing multi-color immunofluorescence experiments with FITC-conjugated KIF17 antibodies, several technical considerations are crucial for optimal results:

First, address spectral considerations: FITC has excitation/emission peaks at approximately 495/519 nm (green spectrum), which must be factored into fluorophore selection for co-staining. Avoid fluorophores with significant spectral overlap such as GFP or Alexa Fluor 488. Instead, select far-red (Alexa Fluor 647), red (Alexa Fluor 594), or blue (DAPI) fluorophores for co-staining experiments. When studying KIF17's association with actin, consider using rhodamine-phalloidin (red) rather than FITC-phalloidin to avoid spectral overlap.

For fixation and permeabilization, paraformaldehyde fixation (4%, 15-20 minutes) preserves KIF17 epitopes while maintaining cellular architecture. Permeabilization with 0.1-0.2% Triton X-100 allows antibody access while preserving subcellular structures where KIF17 localizes. When examining KIF17 at cell-cell junctions, overly harsh permeabilization may disrupt junctional integrity and affect observed localization patterns .

Signal amplification strategies may be necessary as direct FITC conjugation can result in lower signal intensity compared to secondary antibody detection systems. Consider using anti-FITC antibodies conjugated to brighter fluorophores (like Alexa Fluor 488) for signal enhancement if needed.

For co-localization analysis, implement appropriate image acquisition and analysis methods. Acquire z-stacks with optimal step sizes to capture the three-dimensional distribution of KIF17, particularly at cell-cell junctions. Analyze co-localization using Pearson's or Mander's correlation coefficients rather than simple visual overlay to quantitatively assess association with binding partners or cellular structures.

How do I properly analyze and interpret discrepancies between observed molecular weight (170 kDa) and calculated molecular weight (115 kDa) for KIF17 in Western blot analyses?

The discrepancy between KIF17's calculated molecular weight (115 kDa) and its observed migration pattern (170 kDa) in Western blots represents a common challenge in protein research that requires methodical analysis and interpretation. This difference may arise from several factors that should be systematically investigated:

Post-translational modifications significantly impact protein migration patterns. KIF17 may undergo phosphorylation, ubiquitination, SUMOylation, or other modifications that increase its apparent molecular weight. To investigate this possibility, treat protein samples with appropriate enzymes (phosphatases, deubiquitinases) prior to SDS-PAGE to determine if these modifications contribute to the observed migration pattern.

Alternative splicing can result in protein isoforms with different molecular weights. KIF17 has been reported to have splice variants, including KIF17b, which may be recognized by the same antibody . Compare the migration patterns observed in different tissue types, as tissue-specific splicing may occur. Brain tissue, which has been validated for KIF17 Western blot analysis , may express specific isoforms not present in other tissues.

Protein structure and amino acid composition can affect SDS binding and protein migration. Highly acidic or basic regions, or regions rich in proline residues, can cause anomalous migration. To address this, perform Western blot analysis using gradient gels (4-20%) alongside molecular weight markers that span an appropriate range.

For definitive verification, implement complementary approaches: (1) perform mass spectrometry analysis of the 170 kDa band to confirm KIF17 identity and identify potential modifications, (2) compare migration patterns of recombinant KIF17 with endogenous protein, and (3) analyze KIF17 migration in KIF17-depleted or knockout samples to confirm specificity of the observed band.

When presenting Western blot data for KIF17, always include appropriate molecular weight markers and note both the calculated and observed molecular weights to provide proper context for data interpretation.

What are the methodological approaches for studying KIF17 autoinhibition mechanisms in cellular contexts?

Investigating KIF17 autoinhibition requires sophisticated experimental approaches to dissect its dual intramolecular regulatory mechanisms. Based on published research, the following methodological strategies are recommended:

Implement truncation analysis using well-defined KIF17 constructs. Research has established that the C-terminal tail domain (residues 847-1038) is critical for inhibiting microtubule binding, while a coiled-coil segment regulates motility . Systematically express truncated constructs (such as 1-846, 1-738, 1-490, and 1-369) to isolate domain-specific functions . For proper experimental design, verify the dimeric state of each truncation mutant through co-immunoprecipitation of differentially tagged versions (e.g., Myc- and fluorescent protein-tagged) to ensure that observed phenotypes are not due to altered oligomerization .

For visualizing autoinhibition in living cells, employ microtubule-binding assays using AMPPNP treatment. This non-hydrolyzable ATP analog traps kinesin motors in a microtubule-bound state, providing a powerful tool to assess inhibition of microtubule binding. Wild-type KIF17 remains diffuse and cytosolic even after AMPPNP treatment, whereas the tail-truncated 1-846 construct becomes microtubule-bound, demonstrating release from autoinhibition .

To study activation mechanisms, introduce mutations in potential regulatory regions or co-express candidate cargo proteins. Monitor changes in subcellular localization, microtubule binding, and motility using live-cell imaging approaches. For quantitative assessment, combine these cellular assays with in vitro motility assays using purified proteins to directly measure motor activity under defined conditions.

When using antibodies to detect KIF17 in these studies, target epitopes outside the regulatory domains to ensure that antibody binding does not interfere with autoinhibitory mechanisms.

How can I effectively use FITC-conjugated KIF17 antibodies to study its role in epithelial cell polarization and junctional dynamics?

Studying KIF17's role in epithelial polarization and junctional dynamics with FITC-conjugated antibodies requires specialized experimental design and analytical approaches. Based on research findings, implement these methodological strategies:

Utilize both 2D and 3D cell culture systems, as KIF17's functions differ between these contexts. In 2D cultures, KIF17 influences the distribution of actin and E-cadherin, while in 3D organotypic cultures, it regulates actin accumulation at the apical pole . For 3D cultures, embed epithelial cells in Matrigel or collagen matrices and allow cyst formation for 7-10 days before fixation and immunostaining.

For dynamic studies of junctional actin, combine FITC-conjugated KIF17 antibodies with live actin probes. Research has established that co-expression of KIF17 motor domains with fluorescently tagged actin (GFP-actin or mCh-LifeAct) allows visualization of actin incorporation into junctional foci . For fixed samples, apply Sobel edge detection filters to enhance visualization of subtle actin enrichment at cell-cell contacts .

To assess junctional stability, implement calcium depletion assays. Treatment with EGTA or calcium-free media challenges cell-cell adhesions, and recovery upon calcium restoration provides a quantitative measure of junctional stability. KIF17 overexpression has been shown to stabilize cell-cell adhesions against calcium depletion challenges .

For mechanistic studies, combine KIF17 detection with RhoA activity assays, as KIF17 influences RhoA signaling at cell-cell contacts . Use FRET-based RhoA biosensors or RhoA-GTP pulldown assays to quantify active RhoA levels in control versus KIF17-depleted or overexpressing cells. Complement these approaches with inhibitors of RhoA, ROCK, or expression of dominant-negative or constitutively active constructs of RhoA pathway components to establish causality in KIF17-mediated junctional regulation .