FEZ1 Antibody, FITC conjugated

What is FEZ1 protein and why is it a significant research target?

FEZ1 (Fasciculation and Elongation protein zeta-1) is a multifunctional protein that plays crucial roles in axonal outgrowth and is highly expressed in the brain. It serves as a component of the molecular network regulating cellular morphology and axon guidance machinery. FEZ1 has gained significant research interest due to its involvement in multiple cellular processes, including interactions with viral proteins and participation in trafficking pathways. Researchers utilize FITC-conjugated FEZ1 antibodies to visualize and track this protein's subcellular localization, particularly in neuroscience and virology studies. The protein contains multiple functional domains, including acidic poly-glutamate stretches that mediate specific protein-protein interactions, making it an important target for studying neuronal development and viral pathogenesis .

What specific epitope does the FITC-conjugated anti-FEZ1 antibody recognize?

The FITC-conjugated anti-FEZ1 antibody (ABIN7152588) specifically recognizes amino acids 12-200 of human FEZ1 protein. This region encompasses a significant portion of the protein's N-terminal domain, which is known to participate in multiple protein-protein interactions. When designing experiments, researchers should consider that this epitope specificity determines which functional domains of FEZ1 can be detected. The antibody's binding specificity to this region makes it particularly useful for studies focusing on N-terminal interactions rather than the C-terminal coiled-coil region (which falls outside this epitope range). For experiments requiring detection of C-terminal interactions, alternative antibodies targeting different epitopes should be considered .

How does FITC conjugation affect experimental applications compared to unconjugated antibodies?

FITC (fluorescein isothiocyanate) conjugation provides direct fluorescent detection without requiring secondary antibodies, which offers several experimental advantages:

• Streamlined protocols with fewer washing steps and reduced background

• Compatibility with multi-color immunofluorescence using antibodies from the same host species

• Direct quantification in flow cytometry applications

• Reduced cross-reactivity issues encountered with secondary antibodies

What are the optimal fixation and permeabilization conditions when using FITC-conjugated FEZ1 antibody?

For optimal results with FITC-conjugated FEZ1 antibody in immunofluorescence applications:

Fixation options:

• 4% paraformaldehyde (PFA) for 15-20 minutes at room temperature preserves most epitopes while maintaining cellular architecture

• Methanol fixation (100%, -20°C, 10 minutes) may better preserve certain FEZ1 epitopes but can disrupt membrane structures

Permeabilization methods:

• 0.1-0.3% Triton X-100 for 5-10 minutes for standard applications

• 0.05% saponin for gentler permeabilization when studying membrane-associated FEZ1 populations

When studying FEZ1's interactions with kinesin motors or viral capsids, preserving microtubule structures becomes critical, making PFA fixation followed by gentle permeabilization preferable. The epitope recognized by this antibody (AA 12-200) should be accessible with standard permeabilization techniques, but titration of permeabilization conditions may be necessary for optimal signal-to-noise ratio .

How can researchers design co-localization experiments to study FEZ1's interaction with viral capsid proteins?

To effectively study FEZ1 interactions with viral capsid proteins using FITC-conjugated FEZ1 antibody:

• Sample preparation considerations:

  • Synchronize infection to capture specific interaction timepoints

  • Use mild fixation to preserve protein-protein interactions

  • Consider proximity ligation assays for confirming direct interactions

• Microscopy approach:

  • Super-resolution techniques (STED, STORM) can resolve co-localization beyond the diffraction limit

  • Time-lapse imaging with living cells using membrane-permeable dyes for viral particles

• Controls required:

  • FEZ1-depleted cells (siRNA or CRISPR-Cas9) to confirm antibody specificity

  • Competition with recombinant FEZ1 protein to validate binding

  • Non-interacting viral capsid mutants as negative controls

The experimental design should account for FEZ1's established binding to HIV-1 capsid hexamers via its acidic glutamate-rich regions. When analyzing co-localization data, researchers should quantify Pearson's correlation coefficients along microtubule tracks, as FEZ1 functions as a kinesin-1 adaptor protein that facilitates viral trafficking toward the nucleus .

What methodological approaches can reveal the relationship between FEZ1 phosphorylation and its functional interactions?

To investigate how FEZ1 phosphorylation affects its interactions:

• Phosphorylation-specific detection methods:

  • Combine FITC-conjugated FEZ1 antibody with phospho-specific antibodies in dual labeling experiments

  • Use lambda phosphatase treatment as controls to confirm phosphorylation-dependent signals

  • Apply proximity ligation assays to detect interactions only when FEZ1 is in specific phosphorylation states

• Functional validation approaches:

  • Generate phospho-mimetic (S→D/E) and phospho-deficient (S→A) FEZ1 mutants, particularly focusing on Serine 58

  • Perform co-immunoprecipitation with interaction partners using phosphorylation state-specific conditions

  • Implement live-cell FRET biosensors to monitor phosphorylation-dependent interactions in real-time

• Data analysis framework:

  • Quantify co-localization coefficients under various phosphorylation conditions

  • Analyze trafficking velocities of FEZ1-associated cargoes with different phosphorylation states

  • Compare nuclear accumulation rates of viral particles in cells expressing different FEZ1 phospho-variants

Research has established that phosphorylation of FEZ1 at Serine 58 specifically regulates its interaction with kinesin-1 heavy chain, which is required for trafficking and disassembly of HIV-1 capsid during early infection stages. This critical regulatory mechanism should be central to experimental designs investigating FEZ1's transport functions .

What factors contribute to high background when using FITC-conjugated FEZ1 antibody and how can they be mitigated?

High background with FITC-conjugated FEZ1 antibody can result from several factors:

Common causes and solutions:

For neuronal tissues with high autofluorescence, copper sulfate treatment (10mM CuSO₄ in 50mM ammonium acetate) for 10-15 minutes post-fixation can significantly reduce background while preserving FITC signal. Additionally, titrating the antibody concentration is essential, as the optimal working dilution may vary depending on the expression level of FEZ1 in different cell types .

How can researchers validate the specificity of FITC-conjugated FEZ1 antibody signals in their experimental system?

To confirm the specificity of FITC-conjugated FEZ1 antibody signals:

• Genetic validation approaches:

  • Compare staining patterns in FEZ1-knockout cells generated by CRISPR-Cas9

  • Perform siRNA-mediated knockdown with multiple non-overlapping siRNAs

  • Overexpress tagged FEZ1 and confirm co-localization with antibody signal

• Biochemical validation methods:

  • Pre-incubate antibody with recombinant FEZ1 protein (blocking peptide)

  • Compare signals from multiple antibodies targeting different FEZ1 epitopes

  • Conduct western blotting parallel to immunofluorescence to confirm molecular weight

• Cellular and contextual validation:

  • Compare staining patterns with known FEZ1 interaction partners (e.g., HSPA8)

  • Verify expected subcellular localization changes during processes known to affect FEZ1 (e.g., viral infection)

  • Test reactivity in tissues from different species to confirm cross-reactivity claims

Research has demonstrated that FEZ1 depletion induces expression of interferon-stimulated genes, which could serve as an indirect validation of antibody specificity by confirming the functional consequences of FEZ1 knockdown. Multiple independent approaches should be combined to establish robust validation of antibody specificity .

What strategies can enhance detection sensitivity when working with low-abundance FEZ1 in primary cells?

For detecting low-abundance FEZ1 in primary cells:

• Signal amplification methods:

  • Implement tyramide signal amplification (TSA) system compatible with FITC

  • Use biotin-streptavidin systems in combination with FITC-conjugated FEZ1 antibody

  • Consider quantum dot conjugation for improved photostability and brightness

• Sample preparation optimization:

  • Concentrate protein using immunoprecipitation before analysis

  • Optimize antigen retrieval methods (citrate or EDTA-based)

  • Reduce sample thickness for improved signal-to-noise ratio

• Imaging enhancements:

  • Utilize deconvolution algorithms to improve signal resolution

  • Implement photon-counting detectors for weak signals

  • Use spectral unmixing to distinguish FITC signal from autofluorescence

• Protocol refinements:

  • Extend primary antibody incubation to overnight at 4°C

  • Include polyvinyl alcohol or dextran sulfate in antibody diluent to enhance reaction kinetics

  • Use ultrasensitive detection systems (e.g., Alexa Fluor-conjugated anti-FITC antibodies)

For studying FEZ1's neuronal functions, where protein levels may be region-specific, tissue clearing techniques combined with light sheet microscopy can provide enhanced detection sensitivity while preserving the three-dimensional context of FEZ1 distribution .

How can FITC-conjugated FEZ1 antibody be utilized to investigate FEZ1's role in axonal trafficking?

To investigate FEZ1's role in axonal trafficking using FITC-conjugated antibodies:

• Live-cell imaging approaches:

  • Combine with fluorescently tagged cargo proteins in microfluidic chambers

  • Use photoactivatable FITC variants for pulse-chase analysis of FEZ1 movement

  • Apply FRAP (Fluorescence Recovery After Photobleaching) to measure FEZ1 dynamics

• Quantitative analysis frameworks:

  • Track co-movement of FEZ1 with kinesin motors using particle tracking algorithms

  • Measure directional bias (anterograde vs. retrograde) of FEZ1-positive vesicles

  • Quantify velocity profiles and run lengths of FEZ1-associated cargoes

• Experimental manipulations:

  • Apply microtubule-altering drugs to distinguish active transport from diffusion

  • Compare trafficking patterns in neurons expressing FEZ1 phosphorylation mutants

  • Implement optogenetic control of FEZ1 activity using photosensitive protein fusions

Research has established that FEZ1 regulates the balance between retrograde and anterograde motility of HIV-1 particles to ensure net forward movement toward the nucleus. Similar methodology can be applied to study FEZ1's role in trafficking other neuronal cargoes, potentially revealing common mechanisms across different transport processes .

What experimental design would best reveal FEZ1's interactions with retinoic acid receptors (RARs)?

To characterize FEZ1-RAR interactions:

• Protein-protein interaction verification:

  • Combine FITC-conjugated FEZ1 antibody with RAR antibodies in co-immunoprecipitation

  • Use fluorescence anisotropy assays with purified components to measure binding affinities

  • Implement chemical cross-linking followed by mass spectrometry to map interaction interfaces

• Functional analysis approaches:

  • Monitor changes in RAR-responsive element (DR5) binding using FITC-DR5 in the presence/absence of FEZ1

  • Assess effects of all-trans retinoic acid on FEZ1-RAR complex formation

  • Quantify expression of RAR target genes (e.g., hoxb4) in systems with modulated FEZ1 levels

• Cellular localization studies:

  • Track colocalization of FEZ1 and RAR in the perinuclear region during various cell states

  • Investigate changes in complex formation during neuronal differentiation

  • Analyze nuclear translocation patterns with and without retinoic acid treatment

The experimental design should account for the demonstrated increase in hoxb4 gene expression when FEZ1 is overexpressed in the presence of retinoic acid, suggesting functional cooperation between these proteins in transcriptional regulation. Researchers should implement proper controls, including FEZ1 knockdown validation to confirm its role as a hoxb4 inducer .

How can researchers use FITC-conjugated FEZ1 antibody to study its role in antiviral immune responses?

To investigate FEZ1's involvement in antiviral immune responses:

• Expression analysis in immune cells:

  • Monitor FEZ1 localization during viral infection using confocal microscopy

  • Quantify changes in FEZ1 levels in response to interferon treatment

  • Compare FEZ1 distribution patterns between infected and uninfected cells

• Functional interrogation methods:

  • Combine with antibodies against interferon-stimulated genes (ISGs) in dual labeling

  • Track FEZ1-HSPA8 interactions during viral infection and immune activation

  • Measure ISG expression in cells with FEZ1 mutations or deletions

• Mechanistic investigation approaches:

  • Implement time-course analysis of FEZ1 phosphorylation during immune activation

  • Use CRISPR-Cas9 FEZ1 knockout cells to examine effects on innate immune signaling

  • Compare viral particle trafficking in cells with different FEZ1 expression levels

Research has demonstrated that FEZ1 depletion potently suppresses infection by various viruses, including HSV-1, and induces expression of several interferon-stimulated genes (ISGs) such as MxA, MxB, PKR, and ISG56. This suggests a critical role for FEZ1 in regulating antiviral states, independent of the STING pathway. Investigating these mechanisms can provide insights into novel innate immunity regulation pathways .

What methodological approach would best characterize the interaction between FEZ1 and HSPA8?

To characterize the FEZ1-HSPA8 interaction:

• Biochemical interaction analysis:

  • Perform reciprocal co-immunoprecipitations with both FEZ1 and HSPA8 antibodies

  • Use purified components in binding assays to determine direct interaction

  • Implement size exclusion chromatography to characterize complex formation

• Subcellular localization studies:

  • Track co-localization patterns using super-resolution microscopy

  • Monitor redistribution of both proteins during stress responses

  • Examine changes in complex formation during viral infection

• Functional relationship investigation:

  • Compare HSPA8 localization in FEZ1 knockout versus wild-type cells

  • Assess effects of FEZ1 phosphorylation state on HSPA8 binding

  • Investigate the impact of HSPA8 inhibitors on FEZ1-dependent processes

Research has shown that FEZ1 and HSPA8 interact, with even greater enrichment observed for the FEZ1 S58A mutant compared to wild-type. This interaction may be functionally important in regulating FEZ1's role in viral trafficking and innate immune responses. Experimental designs should account for the relatively low enrichment of binding partners compared to input levels, which is expected for highly expressed proteins like HSPA8 that function in multiple cellular processes .

What methodologies can reveal the structural basis for FEZ1's interaction with viral capsid hexamers?

To investigate the structural basis of FEZ1-viral capsid interactions:

• Structural analysis approaches:

  • Utilize cryo-electron microscopy of FEZ1-bound capsid complexes

  • Implement hydrogen-deuterium exchange mass spectrometry to map binding interfaces

  • Apply molecular dynamic simulations to model interaction energetics

• Mutagenesis strategies:

  • Generate point mutations in FEZ1's acidic, poly-glutamate stretches

  • Create capsid mutants with altered central pore electrostatics

  • Develop truncated constructs to identify minimal binding domains

• Competitive binding assays:

  • Test competition between FEZ1 and other known central pore binders (nucleotides, IP6)

  • Measure binding affinities using fluorescence anisotropy with FITC-labeled peptides

  • Quantify binding kinetics using surface plasmon resonance

Research has established that FEZ1 contains multiple acidic, poly-glutamate stretches that interact with the positively charged central pore of CA hexamers. Specifically, residues 182-198 critically contribute to high-affinity CA hexamer binding, and mutation of five sequential glutamates (182EEEEE186 to 182AAAAA186) greatly reduces this interaction. Understanding these structural interactions can provide insights into viral trafficking mechanisms and potential therapeutic targets .

How might FITC-conjugated FEZ1 antibodies be integrated into high-throughput screening approaches?

For integrating FITC-conjugated FEZ1 antibodies into high-throughput screening:

• Assay development considerations:

  • Optimize for microplate format (96/384-well) with automated imaging

  • Develop quantitative readouts for FEZ1 localization or interaction patterns

  • Implement machine learning algorithms for complex phenotype recognition

• Screening applications:

  • Identify small molecules that disrupt FEZ1-viral capsid interactions

  • Screen for compounds that modulate FEZ1 phosphorylation state

  • Discover factors that influence FEZ1's role in immune response regulation

• Validation strategy:

  • Include parallel assays for FEZ1-dependent function (e.g., viral trafficking)

  • Implement dose-response curves for promising hits

  • Develop secondary assays using orthogonal detection methods

High-throughput approaches could reveal novel regulatory mechanisms for FEZ1 function and identify potential therapeutic targets for viral infections that depend on FEZ1 for efficient replication. The multiplexing capability of fluorescence-based detection allows simultaneous monitoring of FEZ1 and its binding partners or downstream effectors .

What novel imaging techniques could enhance our understanding of FEZ1 dynamics in living cells?

Advanced imaging techniques for studying FEZ1 dynamics:

• Super-resolution approaches:

  • STED microscopy to resolve FEZ1 distribution along microtubules below diffraction limit

  • PALM/STORM imaging for single-molecule localization of FEZ1 complexes

  • Expansion microscopy to physically enlarge specimens for enhanced resolution

• Live-cell dynamics visualization:

  • Lattice light-sheet microscopy for 3D tracking with reduced phototoxicity

  • Single-particle tracking with quantum dot-conjugated antibody fragments

  • FRET-FLIM analysis for monitoring FEZ1 conformational changes during function

• Correlative techniques:

  • CLEM (Correlative Light and Electron Microscopy) to visualize FEZ1 in ultrastructural context

  • Cryo-CLEM for capturing FEZ1 complexes in near-native state

  • Super-resolution combined with expansion microscopy for multi-scale analysis

These advanced imaging approaches could reveal the dynamic behavior of FEZ1 during processes such as axonal outgrowth, viral trafficking, and immune response regulation. Particularly valuable would be techniques that allow simultaneous visualization of FEZ1 with its multiple interaction partners in their native cellular environment .