NEK4 Antibody, FITC conjugated

What is NEK4 and why is it a target of interest in research?

NEK4 (NIMA-related kinase 4) is a serine/threonine protein kinase that acts primarily on threonine residues. It belongs to the NEK family of kinases and has emerged as an important regulatory protein in multiple cellular processes. NEK4 is of significant research interest due to its involvement in DNA damage response pathways, cilia maintenance, microtubule stabilization, apoptosis signaling, stress response, translation, protein quality control, and RNA splicing regulation . Recent interactome studies have revealed that NEK4 interacts with proteins in various cellular compartments including the nucleus, mitochondria, and endoplasmic reticulum, suggesting its diverse functional roles .

What are the known isoforms of NEK4 and how do they differ?

Two main isoforms of NEK4 have been characterized: NEK4.1 (isoform 1) and NEK4.2 (isoform 2). The primary structural difference between these isoforms is that NEK4.1 contains a 138 bp insertion in its regulatory domain compared to NEK4.2 . This structural difference translates to significant functional divergence - NEK4.1 has approximately three times more interaction partners (474) than NEK4.2 (150), with only about 102 proteins common to both isoforms . While both isoforms participate in some common processes such as mRNA splicing, apoptosis, and cell cycle checkpoint regulation, NEK4.1 appears to have additional functions in DNA repair and cilia processes that are not observed with NEK4.2 .

How does FITC conjugation affect NEK4 antibody application and detection sensitivity?

FITC (fluorescein isothiocyanate) conjugation to NEK4 antibodies provides direct fluorescent detection capabilities without requiring secondary antibodies. The conjugation process typically involves binding FITC to the primary amines in the side chains of lysine residues and the N-terminus of the antibody using standard chemical labeling methods . While FITC conjugation enables direct visualization in applications like flow cytometry and immunofluorescence microscopy, researchers should be aware that the conjugation process may slightly reduce antibody affinity in some cases. The excitation maximum of FITC is approximately 495 nm and emission maximum around 520 nm, providing a bright green fluorescence suitable for most standard fluorescence detection systems.

What are the optimal conditions for using FITC-conjugated NEK4 antibodies in immunofluorescence studies?

For optimal immunofluorescence results with FITC-conjugated NEK4 antibodies, researchers should consider several critical parameters:

• Fixation method: Paraformaldehyde (4%) fixation for 15-20 minutes at room temperature is generally recommended for preserving NEK4 antigenicity while maintaining cellular structure.

• Permeabilization: Use 0.1-0.2% Triton X-100 for 5-10 minutes to allow antibody access to intracellular NEK4.

• Blocking: A 1-hour incubation with 5% normal serum (matched to secondary antibody host if using additional non-conjugated primary antibodies) with 0.1% BSA helps reduce background fluorescence.

• Antibody dilution: Start with manufacturer recommended dilutions (typically 1:50-1:200) and optimize based on signal-to-noise ratio.

• Incubation conditions: Overnight incubation at 4°C generally yields better results than shorter incubations at room temperature.

• Photobleaching prevention: Mount slides using anti-fade mounting media containing DAPI for nuclear counterstaining while minimizing FITC photobleaching.

• Controls: Include a negative control using an isotype-matched FITC-conjugated antibody to assess non-specific binding.

NEK4 shows predominantly nuclear localization but has also been detected in mitochondrial fractions , so appropriate subcellular markers should be considered for colocalization studies.

How can researchers validate the specificity of FITC-conjugated NEK4 antibodies?

Validating antibody specificity is crucial for generating reliable data. For FITC-conjugated NEK4 antibodies, a multi-step validation approach is recommended:

• Western blot analysis: Perform parallel Western blot with the non-conjugated antibody from the same clone to verify that a single band of expected molecular weight (approximately 95 kDa for NEK4) is detected.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before immunostaining to confirm that the signal is specifically blocked.

• Genetic validation: Use cells with NEK4 knockdown (siRNA or CRISPR-Cas9) or overexpression systems to confirm corresponding decrease or increase in signal intensity.

• Isoform specificity testing: If distinguishing between NEK4.1 and NEK4.2, validate using recombinant proteins or cells expressing only one isoform to confirm the antibody's ability to recognize the intended target.

• Cross-reactivity assessment: Test the antibody on cells or tissues from different species if cross-species reactivity is claimed by the manufacturer.

• Comparison with alternative NEK4 antibodies: Comparing staining patterns with antibodies targeting different epitopes of NEK4 can further validate specificity.

What are the recommended protocols for using FITC-conjugated NEK4 antibodies in flow cytometry?

For optimal flow cytometry results with FITC-conjugated NEK4 antibodies:

• Cell preparation:

  • Harvest cells using gentle enzymatic detachment to preserve surface proteins

  • Fix with 2-4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilize with 0.1% saponin or 0.1% Triton X-100 for intracellular NEK4 detection

• Staining protocol:

  • Resuspend cells at 1 × 10^6 cells/100 μL in staining buffer (PBS with 0.5% BSA)

  • Block with 2% normal serum for 20 minutes

  • Add FITC-conjugated NEK4 antibody at manufacturer-recommended concentration (typically 2-10 μg/mL)

  • Incubate for 30-60 minutes at room temperature in the dark

  • Wash twice with 2 mL staining buffer

  • Resuspend in 400-500 μL buffer for analysis

• Controls:

  • Unstained cells for autofluorescence baseline

  • Isotype control conjugated to FITC at the same concentration

  • Single-color controls if performing multicolor analysis

• Instrument settings:

  • Use 488 nm laser for FITC excitation

  • Collect emission through a 530/30 nm bandpass filter

  • Adjust voltage settings based on negative control samples

  • Compensate for spectral overlap if using multiple fluorophores

Published data indicates that anti-NEK4 CAR-293 cells stained with 100 μL of 10 μg/mL FITC-labeled human NEK4 can be used to evaluate binding activity in flow cytometry applications .

How can researchers distinguish between NEK4 isoforms in experimental analysis?

Distinguishing between NEK4.1 and NEK4.2 isoforms requires careful experimental design:

• Antibody selection: Use isoform-specific antibodies that target the 138 bp insertion region present in NEK4.1 but absent in NEK4.2. For the FITC-conjugated antibodies, verify whether they target a common region (like the kinase domain) or an isoform-specific region.

• RT-PCR analysis: Design primers that flank the insertion region to identify both isoforms simultaneously based on PCR product size differences.

• Western blot identification: NEK4.1 (~95 kDa) should show a slightly higher molecular weight compared to NEK4.2 (~90 kDa) due to the 46 amino acid insertion.

• Functional assays: Based on research by Basei et al., researchers can design splicing reporter assays, as the two isoforms show opposing effects on RNA splicing regulation - NEK4.2 demonstrates preference for distal splicing sites while NEK4.1 kinase dead mutant increases proximal splice site selection .

• Interactome analysis: Perform immunoprecipitation followed by mass spectrometry, as NEK4.1 and NEK4.2 have distinct interaction partners. Only NEK4.2 interacts with splicing regulators like SRSF1, SRSF2, and SRPK1, while both interact with hnRNPA1 .

• Subcellular localization: While both isoforms show predominantly nuclear localization, performing detailed colocalization studies with known partners unique to each isoform can help distinguish their specific cellular distributions.

What are common causes of background fluorescence when using FITC-conjugated NEK4 antibodies, and how can they be mitigated?

Background fluorescence can significantly impact the interpretation of results using FITC-conjugated antibodies. Common causes and solutions include:

• Non-specific antibody binding:

  • Mitigation: Increase blocking time (1-2 hours) with 5-10% normal serum and optimize antibody concentration through titration experiments.

  • Solution: Add 0.1-0.3% Triton X-100 to blocking buffer to reduce hydrophobic interactions.

• Cell/tissue autofluorescence:

  • Mitigation: Include unstained samples as controls and consider using Sudan Black B (0.1-0.3% in 70% ethanol) incubation for 10-20 minutes after antibody staining to quench autofluorescence.

  • Solution: Use narrow bandpass filters on microscopes/flow cytometers to reduce detection of autofluorescence, which typically has a broader emission spectrum.

• Fixative-induced fluorescence:

  • Mitigation: Minimize fixation time and wash thoroughly after fixation (3-5 washes).

  • Solution: Consider using methanol fixation instead of aldehydes when compatible with the epitope.

• Photobleaching during analysis:

  • Mitigation: Use anti-fade mounting media containing agents like DABCO or PPD.

  • Solution: Minimize exposure time during imaging and analyze samples promptly after staining.

• FITC-specific issues:

  • Mitigation: FITC is pH-sensitive and works best at pH 8.0-9.0; ensure buffers are properly pH-adjusted.

  • Solution: Consider alternative green fluorophores like Alexa Fluor 488 if persistent problems occur with FITC.

• Excess unbound FITC:

  • Mitigation: Ensure FITC-conjugated antibodies are properly purified with no free FITC remaining.

  • Solution: Dialyze the antibody preparation if free FITC contamination is suspected.

What controls should be included when performing colocalization studies with FITC-conjugated NEK4 antibodies?

Robust colocalization studies with FITC-conjugated NEK4 antibodies require multiple controls:

• Single-labeling controls:

  • Samples labeled with only FITC-conjugated NEK4 antibody to assess bleed-through

  • Samples labeled with only the second marker to assess bleed-through in reverse

• Antibody controls:

  • Isotype control for FITC-conjugated NEK4 antibody

  • Secondary-only control for the co-staining marker

• Biological controls:

  • NEK4 knockdown/knockout cells to confirm specificity

  • Positive control cells known to express NEK4 and the co-marker of interest

• Processing controls:

  • Parallel processing of all samples to ensure consistent staining conditions

  • No-primary antibody controls to assess secondary antibody non-specific binding

• Imaging controls:

  • Sequential acquisition of channels to minimize bleed-through

  • Consistent exposure settings across all samples

• Analysis controls:

  • Use appropriate colocalization coefficients (Pearson's, Manders')

  • Apply threshold controls determined by single-stained samples

  • Include random colocalization controls (images rotated 90° or flipped)

When studying NEK4 subcellular localization, appropriate markers should be selected based on expected localization patterns. For instance, when investigating mitochondrial localization, ANT3 can be used as a mitochondrial marker based on previous studies , though the colocalization may be partial rather than complete.

How can FITC-conjugated NEK4 antibodies be utilized to study the role of NEK4 in DNA damage response pathways?

FITC-conjugated NEK4 antibodies can be powerful tools for investigating NEK4's role in DNA damage response through several advanced approaches:

• Live cell imaging of DNA damage dynamics:

  • Transfect cells with fluorescently-tagged DNA damage markers (e.g., 53BP1-RFP)

  • Microinject or transduce cell-permeable FITC-NEK4 antibodies

  • Monitor real-time recruitment and dissociation of NEK4 at damage sites using confocal microscopy

• DNA damage-specific phosphorylation analysis:

  • Treat cells with DNA-damaging agents (e.g., etoposide, UV, ionizing radiation)

  • Fix and stain with FITC-NEK4 antibodies alongside phospho-specific antibodies for DNA repair factors

  • Quantify changes in NEK4 localization and potential phosphorylation state changes

• Laser microirradiation studies:

  • Subject cells to focused laser microirradiation to induce localized DNA damage

  • Track NEK4 recruitment using FITC-conjugated antibodies in fixed cells at different time points

  • Compare recruitment kinetics with known DNA repair factors

• Functional knockdown/rescue experiments:

  • Deplete endogenous NEK4 using siRNA or CRISPR

  • Rescue with wild-type or mutant NEK4 variants

  • Use FITC-NEK4 antibodies to quantify expression levels and localization patterns

  • Correlate with DNA repair efficiency measured by comet assay or γH2AX foci resolution

• Interaction partner analysis during DNA damage:

  • Leverage the known interaction between NEK4 and the DNA repair protein Ku70 (XRCC6)

  • Perform proximity ligation assays combining FITC-NEK4 antibodies with Ku70 antibodies

  • Quantify interaction frequency under different DNA damage conditions

Research has shown that Ku70 phosphorylation levels vary between cells expressing wild-type NEK4.1 and those expressing kinase-dead mutants, suggesting that NEK4 may directly or indirectly regulate Ku70 phosphorylation status during DNA repair processes .

How can researchers investigate the differential functions of NEK4 isoforms using FITC-conjugated antibodies?

Investigating the functional differences between NEK4.1 and NEK4.2 requires sophisticated experimental designs:

• Isoform-specific knockdown-rescue systems:

  • Design siRNAs targeting the unique 138 bp insertion region in NEK4.1

  • Rescue with siRNA-resistant constructs of either isoform

  • Use FITC-conjugated antibodies to validate expression and localization

• RNA splicing functional assays:

  • Utilize the E1A reporter system that demonstrated differential effects of NEK4 isoforms on splicing site selection

  • NEK4.2 promotes distal splicing sites (13S formation), while NEK4.1 kinase dead mutant increases proximal site usage (9S formation)

  • Couple with FITC-conjugated antibody staining to correlate expression levels with splicing effects

• Interactome comparative analysis:

  • Immunoprecipitate each isoform separately under identical conditions

  • Identify differential binding partners through mass spectrometry

  • Validate key interactions using FITC-conjugated NEK4 antibodies in proximity ligation assays

• Cellular phenotype rescue experiments:

  • In cells depleted of endogenous NEK4, introduce individual isoforms

  • Measure functional outcomes such as:

    • Cell cycle progression

    • DNA damage repair efficiency

    • Primary cilia formation

    • RNA splicing patterns

  • Use FITC-conjugated antibodies to quantify expression levels

• Post-translational modification profiling:

  • Immunoprecipitate phosphorylated proteins from cells expressing either NEK4 isoform

  • Compare phosphorylation profiles of specific targets like Ku70

  • Use FITC-conjugated antibodies for quantification in parallel samples

Research has already demonstrated that while both isoforms interact with hnRNPA1, only NEK4.2 interacts with other splicing regulators like SRSF1, SRSF2, and SRPK1, potentially explaining their differential effects on RNA splicing regulation .

What are the recent advances in using FITC-conjugated NEK4 antibodies for studying primary cilia formation and maintenance?

Recent advances in studying NEK4's role in cilia biology using FITC-conjugated antibodies include:

• Super-resolution microscopy applications:

  • Using STORM or STED microscopy with FITC-NEK4 antibodies to precisely localize NEK4 within ciliary structures

  • Combining with ciliary markers like acetylated tubulin or IFT proteins for nanoscale colocalization analysis

  • Quantifying spatial relationships between NEK4 and its interaction partners like Whirlin in ciliary regions

• Ciliopathy model investigations:

  • Comparing NEK4 localization patterns in cells derived from ciliopathy patients versus healthy controls

  • Correlating NEK4 expression levels and distribution with ciliary phenotypes

  • Using FITC-NEK4 antibodies in high-throughput screening for therapeutic compounds that normalize NEK4 distribution

• Intraflagellar transport (IFT) dynamics:

  • Using FITC-NEK4 antibodies alongside markers for IFT proteins identified as NEK4 interactors (TTC21B and IFT172)

  • Time-course fixation studies following ciliary induction to track NEK4 recruitment

  • Correlating NEK4 levels with ciliary growth rates and maintenance

• Ciliary signaling pathway integration:

  • Investigating NEK4's relationship with other ciliary proteins like RPGRIP1L (Fanton), a known NEK4 interaction partner

  • Using FITC-NEK4 antibodies in combination with pathway-specific markers

  • Analyzing changes in NEK4 localization in response to pathway stimulation or inhibition

• Comparative analysis of NEK4 isoforms in cilia:

  • Using isoform-specific antibodies or FITC-conjugated general NEK4 antibodies with isoform-specific overexpression

  • Quantifying the relative contributions of each isoform to ciliary functions

  • Correlating with interactome data showing differential binding to ciliary proteins

Research has demonstrated that NEK4 colocalizes with Whirlin, which itself colocalizes with RPGR ORF15 in photoreceptor connecting cilia , suggesting a potential role for NEK4 in specialized ciliary functions that could be further explored using FITC-conjugated antibodies.

What quality control measures should researchers implement when using FITC-conjugated NEK4 antibodies?

Comprehensive quality control for FITC-conjugated NEK4 antibodies should include:

• Fluorophore-to-protein ratio assessment:

  • Determine the F/P ratio using spectrophotometric analysis

  • Optimal F/P ratios usually range from 2:1 to 6:1 for FITC conjugates

  • Higher ratios may cause quenching and reduced antibody activity

• Lot-to-lot consistency validation:

  • Compare new antibody lots with previously validated lots

  • Test using consistent positive control samples

  • Document sensitivity and specificity parameters across lots

• Spectral profile verification:

  • Measure excitation/emission spectra to confirm proper FITC conjugation

  • Verify peak excitation at ~495 nm and emission at ~520 nm

  • Check for any unexpected spectral shifts that might indicate improper conjugation

• Stability assessment:

  • Test antibody performance after different storage durations

  • Evaluate freeze-thaw stability through multiple cycles

  • Monitor for signs of aggregation or precipitation

• Application-specific validation:

  • For flow cytometry: Verify consistent staining index across experiments

  • For microscopy: Assess signal-to-noise ratio and photobleaching resistance

  • For quantitative applications: Establish standard curves with recombinant NEK4 protein

• Cross-reactivity profile:

  • Test against cell lines with variable NEK4 expression levels

  • Verify specificity across multiple human cell types

  • Confirm expected subcellular localization patterns

Available products like FITC-labeled human NEK4 protein (His-tagged) can be used as positive controls, as they have been QC tested for binding to immobilized anti-NEK4 antibody with a linear range of 10-78 ng/mL .

How does epitope selection affect the performance of FITC-conjugated NEK4 antibodies?

Epitope selection significantly impacts antibody performance across applications:

• Domain-specific targeting considerations:

  • Kinase domain epitopes (N-terminal region): May detect both active and inactive forms but might not distinguish between isoforms

  • Regulatory domain epitopes (C-terminal region): May provide isoform specificity due to the 138 bp insertion in NEK4.1

  • Available FITC-conjugated antibodies targeting amino acids 516-661 of human NEK4 target the C-terminal region

• Functional impact assessment:

  • Antibodies targeting the kinase domain may interfere with enzymatic activity

  • Antibodies targeting protein-protein interaction regions may disrupt NEK4 complexes

  • C-terminal targeting antibodies may be preferable for detecting native protein complexes

• Post-translational modification considerations:

  • Phosphorylation-sensitive epitopes: May give different signals depending on NEK4 activation state

  • Epitopes near NLS (nuclear localization signal): May affect nuclear transport or detection in different cellular compartments

  • Epitopes near protein-protein interaction domains: May be masked in certain protein complexes

• Structural accessibility analysis:

  • Surface-exposed epitopes provide better detection in native applications

  • Epitopes in flexible regions may be more accessible but potentially less specific

  • Conserved vs. variable region targeting affects species cross-reactivity

• Application-specific epitope selection:

  • For detecting active NEK4: Target phosphorylation-specific epitopes

  • For isoform discrimination: Target the 138 bp insertion region unique to NEK4.1

  • For interaction studies: Target regions away from known protein-binding domains

Currently available FITC-conjugated anti-NEK4 antibodies targeting AA 516-661 are suitable for detecting human NEK4, but researchers should carefully consider the experimental context when selecting the most appropriate epitope target.

What are the technical considerations for multiplexing FITC-conjugated NEK4 antibodies with other fluorophore-conjugated antibodies?

Successful multiplexing with FITC-conjugated NEK4 antibodies requires careful technical considerations:

• Spectral compatibility planning:

  • FITC (Ex: 495 nm, Em: 520 nm) pairs well with:

    • Red fluorophores: PE (Ex: 565 nm, Em: 578 nm) or APC (Ex: 650 nm, Em: 660 nm)

    • Far-red fluorophores: Cy5 (Ex: 650 nm, Em: 670 nm) or Alexa Fluor 647 (Ex: 650 nm, Em: 668 nm)

    • Blue fluorophores: Pacific Blue (Ex: 401 nm, Em: 452 nm)

  • Avoid: PE-Cy5.5, which has spectral overlap with FITC

• Protocol optimization strategies:

  • Sequential staining: Apply FITC-NEK4 antibody first, fix lightly, then apply additional antibodies

  • Buffer compatibility: Ensure all antibodies perform optimally in the same buffer system

  • Incubation timing: Optimize incubation periods that work for all antibodies in the panel

• Compensation and controls:

  • Single-stained controls for each fluorophore

  • Fluorescence-minus-one (FMO) controls

  • Isotype controls for each antibody class and fluorophore combination

  • Unstained controls for autofluorescence baseline

• Cross-blocking assessment:

  • Test for epitope competition between antibodies targeting related proteins

  • Verify that antibody binding is not sterically hindered in multiplexed panels

  • Establish optimal antibody concentration ratios

• Instrument setup considerations:

  • Use appropriate laser combinations (488 nm for FITC)

  • Apply narrow bandpass filters to minimize spectral overlap

  • Perform mathematical compensation to correct for any remaining spillover

• Fixation compatibility:

  • Ensure selected fixation method preserves all epitopes

  • Test fixation impact on each fluorophore's signal intensity

  • Consider differential fixation approaches if needed

For studying NEK4's involvement in DNA damage response or cilia formation, multiplexing with DNA repair markers (γH2AX, 53BP1) or ciliary markers (acetylated tubulin, IFT88) respectively can provide valuable contextual information when analyzed alongside FITC-conjugated NEK4 antibodies.

Human subjects are governed by protocols that should be approved by appropriate ethical committees, and all experiments should be performed in accordance with relevant guidelines and regulations.