NEK4 Antibody, Biotin conjugated

What is NEK4 and why is it important in cellular research?

NEK4 (Never in mitosis A-related kinase 4) is a serine/threonine-protein kinase belonging to the NEK family of protein kinases. It plays significant roles in cell cycle regulation, cell division, mitosis, and protein phosphorylation processes . As part of the wider NEK family, NEK4 contributes to critical cellular functions including cilium assembly and potentially DNA damage responses, similar to other NEK proteins such as NEK1 . Understanding NEK4's functions provides insights into fundamental cellular processes and potential disease mechanisms, making it a valuable target for research in both normal cellular physiology and pathological conditions.

What are the key specifications of commercially available NEK4 antibody, biotin conjugated?

The typical commercially available NEK4 antibody with biotin conjugation has the following specifications:

• Generated using recombinant human serine/threonine-protein kinase NEK4 protein (amino acids 516-661) as the immunogen

• Produced in rabbit hosts, resulting in polyclonal IgG antibodies

• Purified using Protein G purification methods with >95% purity

• Stored in buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as preservative

• Recommended storage at -20°C or -80°C to maintain stability

• Primary tested application in ELISA, though may be suitable for other applications depending on validation

How does NEK4 relate to other members of the NEK kinase family?

NEK4 belongs to Group 1 of the NEK kinase family based on substrate specificity profiling. This grouping also includes NEK1 and NEK3, characterized by their shared preference for arginine in the -1 position of substrate peptides . Within this group, NEK4 shows strong selectivity for lysine and arginine in the +2 position and lacks preference for tryptophan in the -3 position, which distinguishes it from some other NEK family members .

The NEK family can be categorized into four specificity groups:

• Group 1: NEK1, NEK3, NEK4 (preference for arginine at -1 position)

• Group 2: NEK5, NEK8 (less prominent selection for arginine at -1)

• Group 3: NEK2, NEK10 (serine phospho-acceptor specificity)

• Group 4: NEK6, NEK7, NEK9 (preference for acidic residues at -5, -4, -2 positions)

Understanding these relationships helps researchers interpret NEK4 findings in the broader context of NEK family function.

What are the optimal experimental conditions for using biotin-conjugated NEK4 antibodies in proximity labeling studies?

Biotin-conjugated antibodies like the NEK4 antibody can be effectively used in proximity labeling methods such as Biotinylation by Antibody Recognition (BAR). For optimal results when adapting BAR methodology for NEK4 studies, researchers should:

• Begin with properly fixed and permeabilized samples to ensure antibody accessibility while preserving native protein interactions

• Use validated primary anti-NEK4 antibodies followed by HRP-conjugated secondary antibodies

• Perform biotinylation reaction with hydrogen peroxide and phenol biotin at appropriate concentrations

• Optimize reaction time to balance specific biotinylation against background labeling

• Use stringent washing and harsh solubilization conditions after biotinylation is complete

• Capture biotinylated proteins with streptavidin-coated beads for subsequent analysis

This approach allows researchers to identify proteins in close proximity to NEK4 in various cellular contexts, including primary tissues where traditional fusion protein approaches are not feasible.

How can substrate specificity information guide the design of NEK4 functional studies?

NEK4's substrate specificity profile provides crucial information for designing functional studies. Based on comprehensive substrate profiling data, NEK4 shows distinctive preferences that can guide experimental design:

• Strong selection for arginine in the -1 position relative to the phosphorylation site

• Preference for lysine and arginine in the +2 position

• Notable lack of preference for tryptophan in the -3 position, unlike many other NEK kinases

Researchers can use these specificity determinants to:

• Design optimal peptide substrates for in vitro kinase assays

• Create prediction algorithms to identify potential physiological NEK4 substrates

• Engineer phosphorylation site mutants to test NEK4-dependent regulation

• Develop NEK4-specific kinase inhibitors based on substrate recognition features

Understanding these specificity determinants is particularly valuable when investigating the distinct cellular roles of NEK4 compared to other closely related kinases like NEK1 and NEK3.

What considerations should be made when comparing results from different NEK4 antibody formats?

When comparing results obtained using different formats of NEK4 antibodies (such as biotin-conjugated versus unconjugated), researchers should consider several important factors:

• Epitope accessibility: Biotin conjugation might affect epitope recognition, potentially altering binding affinity or specificity. Cross-validation with multiple antibodies recognizing different epitopes is recommended.

• Background signals: Biotin-conjugated antibodies may generate different background patterns compared to unconjugated versions, particularly in tissues with endogenous biotin. Pre-blocking endogenous biotin is essential for accurate results.

• Detection system compatibility: While biotin-conjugated antibodies work excellently with streptavidin-based detection systems, they may not be optimal for all experimental platforms.

• Steric hindrance: The biotin moiety might cause steric hindrance in crowded cellular environments, potentially affecting antibody performance in certain applications.

• Storage and stability differences: Conjugated antibodies may have different stability profiles compared to unconjugated versions .

Researchers should perform parallel validation experiments when transitioning between antibody formats to ensure consistency of results.

How can biotin-conjugated NEK4 antibodies be effectively used in proximity labeling experiments?

Biotin-conjugated NEK4 antibodies can be integrated into proximity labeling experiments using the BAR method to identify protein-protein interactions involving NEK4. The methodological approach involves:

• Sample preparation:

  • Fix cells or tissue samples with an appropriate fixative (typically formaldehyde)

  • Permeabilize samples to allow antibody penetration while maintaining protein interactions

• Primary antibody application:

  • Apply the anti-NEK4 primary antibody at optimized concentration

  • Incubate at 4°C overnight to ensure thorough binding

• Secondary antibody and biotinylation:

  • Apply HRP-conjugated secondary antibody

  • Initiate biotinylation reaction with hydrogen peroxide and phenol biotin

  • Control reaction time carefully to minimize non-specific labeling

• Protein isolation and analysis:

  • Reverse cross-linking and solubilize proteins under harsh conditions

  • Capture biotinylated proteins using streptavidin-coated beads

  • Identify interacting proteins via mass spectrometry

This approach offers the significant advantage of identifying NEK4 interactors in their native cellular context, including in primary tissues where traditional approaches like BioID or APEX2 cannot be easily applied.

What are the optimal storage and handling conditions for maintaining NEK4 antibody, biotin conjugated activity?

To maintain optimal activity of biotin-conjugated NEK4 antibodies, researchers should follow these storage and handling guidelines:

• Long-term storage:

  • Store at -20°C or preferably -80°C in small aliquots

  • Avoid repeated freeze-thaw cycles which can degrade antibody activity

• Working solution preparation:

  • Thaw aliquots rapidly at room temperature or 37°C

  • Mix gently by inversion rather than vortexing to avoid protein denaturation

• Buffer conditions:

  • The presence of 50% glycerol in the storage buffer helps maintain stability

  • The pH 7.4 PBS buffer provides optimal conditions for antibody stability

  • 0.03% Proclin 300 preservative prevents microbial contamination

• Handling precautions:

  • Avoid prolonged exposure to light which can affect the biotin conjugate

  • Work with clean pipettes and tubes to prevent contamination

  • Consider adding carrier proteins for very dilute working solutions

Following these guidelines will help ensure consistent antibody performance across experiments and maximize the shelf-life of these valuable reagents.

What control experiments should be included when using NEK4 antibody, biotin conjugated?

Rigorous control experiments are essential when using biotin-conjugated NEK4 antibodies to ensure data reliability. Researchers should include:

• Antibody specificity controls:

  • Immunoprecipitation followed by western blotting to confirm antibody specificity

  • Pre-absorption with immunogen peptide to validate specific binding

  • Parallel experiments with non-targeting control IgG of the same species

• Biotin conjugation controls:

  • Unconjugated primary antibody with separate biotinylation step

  • No-primary-antibody control to assess non-specific biotinylation

  • Endogenous biotin blocking to prevent false-positive signals

• Experimental validation controls:

  • Known NEK4 interacting partners as positive controls

  • Spatially separated proteins as negative controls

  • Kinase-dead mutants when studying phosphorylation events

• For proximity labeling experiments:

  • Omission of hydrogen peroxide to control for non-enzymatic biotinylation

  • Samples with no phenol biotin to establish background signal levels

  • Various reaction time points to optimize signal-to-noise ratio

Implementing these controls will help distinguish genuine NEK4-related signals from background and ensure reproducible, interpretable results.

How can researchers address non-specific binding when using NEK4 antibody, biotin conjugated?

Non-specific binding is a common challenge when using biotin-conjugated antibodies. To minimize this issue with NEK4 antibodies:

• Optimize blocking conditions:

  • Use a combination of BSA or serum with non-ionic detergents

  • Consider specialized blocking reagents for biotin-streptavidin systems

  • Block endogenous biotin using avidin or streptavidin pre-treatment

• Adjust antibody concentration:

  • Titrate antibody to determine optimal concentration

  • Lower concentrations may reduce non-specific binding while maintaining specific signals

• Modify washing procedures:

  • Increase wash duration and/or frequency

  • Adjust salt concentration or detergent type in wash buffers

  • Consider additives like polyethylene glycol to reduce non-specific interactions

• Sample preparation refinements:

  • Optimize fixation to preserve epitopes while reducing background

  • Consider alternative permeabilization methods if membrane proteins show high background

• Validate results with alternative methods:

  • Confirm key findings using non-biotinylated antibodies

  • Use complementary techniques like co-immunoprecipitation to validate interactions

Implementing these strategies systematically can significantly improve signal-to-noise ratio in experiments using biotin-conjugated NEK4 antibodies.

What statistical approaches are most appropriate for analyzing NEK4 proximity labeling data?

Proximity labeling experiments with NEK4 antibodies generate complex datasets that require robust statistical analysis. Recommended approaches include:

• For label-free quantification:

  • Use fold-change and p-value cutoffs based on replicate experiments

  • Apply SAINT (Significance Analysis of INTeractome) algorithm for scoring interactions

  • Consider probabilistic scoring methods that account for protein abundance

• For SILAC or TMT-based quantification:

  • Calculate SILAC or TMT ratios and apply appropriate normalization

  • Use statistical tests appropriate for the experimental design (t-tests for simple comparisons, ANOVA for multiple conditions)

  • Apply multiple testing corrections such as Benjamini-Hochberg FDR

• Data visualization and exploration:

  • Generate volcano plots showing fold-change versus significance

  • Use hierarchical clustering to identify protein groups with similar behavior

  • Apply principal component analysis to identify major sources of variation

• Functional analysis:

  • Perform Gene Ontology enrichment analysis on identified interactors

  • Map identified proteins to known protein complexes or pathways

  • Use protein-protein interaction databases to extend network analysis

These approaches help distinguish genuine NEK4 interactors from background proteins and provide biological context for interpreting results.

How can researchers integrate NEK4 substrate specificity data with proximity labeling results?

Integrating substrate specificity information with proximity labeling results can provide deeper insights into NEK4 biology:

• Bioinformatic integration:

  • Scan proximity-labeled proteins for NEK4 consensus motifs (preference for arginine at -1 position, lysine/arginine at +2)

  • Prioritize proteins that are both proximal to NEK4 and contain potential phosphorylation sites

  • Calculate enrichment scores for consensus motifs in the proximity dataset

• Experimental validation:

  • Design phosphorylation site-specific antibodies for candidate substrates

  • Perform in vitro kinase assays with recombinant NEK4 and candidate substrates

  • Create phosphorylation site mutants to test functional relevance

• Differential analysis:

  • Compare proximity labeling results from wild-type versus kinase-dead NEK4

  • Identify proteins that change association upon cellular stimulation

  • Correlate changes in proximity with changes in phosphorylation state

• Network analysis:

  • Construct integrated networks incorporating known NEK4 substrates and newly identified proximity partners

  • Map kinase-substrate relationships within larger protein complexes

  • Identify potential scaffolding proteins that might coordinate NEK4 with its substrates

This integrative approach builds a more complete picture of NEK4 function by connecting its enzymatic activity with its protein interaction network.

How does the substrate specificity of NEK4 compare to other NEK family members?

NEK4's substrate specificity exhibits both similarities and differences compared to other NEK family members:

Key points about NEK4's specificity include:

• NEK4 shows a distinctive lack of preference for tryptophan in the -3 position, unlike other NEK kinases

• NEK4 demonstrates strong selection for positively charged residues (Arg/Lys) at the +2 position

• NEK4 shares substrate preferences with NEK1, but each likely has unique cellular roles based on expression patterns and additional interacting partners

Understanding these specificity differences helps researchers design specific assays and identify the most likely physiological substrates for each NEK family member.

What emerging technologies might enhance NEK4 interaction studies beyond current approaches?

Several emerging technologies hold promise for advancing NEK4 interaction studies:

• Proximity-dependent technologies:

  • TurboID and miniTurbo for faster biotin labeling with lower background

  • Split-TurboID to study condition-dependent protein-protein interactions

  • APEX2-based proximity labeling with millisecond temporal resolution

• Advanced imaging approaches:

  • Super-resolution microscopy combined with proximity labeling

  • Live-cell imaging of NEK4 interactions using split fluorescent proteins

  • Correlative light and electron microscopy to study NEK4 localization at ultrastructural level

• Proteomics innovations:

  • Crosslinking mass spectrometry to map specific interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to study interaction dynamics

  • Thermal proteome profiling to identify NEK4-dependent complexes

• Genetic approaches:

  • CRISPR-based screening to identify functional NEK4 interactors

  • Proximity-dependent genetic systems like APEX-Peroxidase Assisted Capture-Transcription

  • Base editing to introduce specific mutations in NEK4 or interacting partners

These technologies, when adapted for studying NEK4, will likely provide unprecedented insights into its dynamic interactions and cellular functions.