NEK4 Antibody, HRP conjugated

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

NEK4 is a serine/threonine protein kinase belonging to the NIMA-related kinase family that plays crucial roles in multiple cellular processes. It is required for normal entry into proliferative arrest after a limited number of cell divisions (replicative senescence) and for normal cell cycle arrest in response to double-stranded DNA damage . Recent studies have also implicated NEK4 in epithelial-mesenchymal transition (EMT) regulation, which promotes cancer metastasis . Additionally, NEK4 functions in cilia maintenance, microtubule stabilization, and most intriguingly, RNA splicing control . This multifunctional nature makes NEK4 a significant target in various research contexts, from basic cell biology to cancer research.

What are the key applications for NEK4 antibodies in research?

NEK4 antibodies, particularly HRP-conjugated variants, serve multiple experimental purposes in research settings:

• ELISA applications (recommended dilution 1:500-1:1000) for protein quantification and interaction studies

• Western blotting for detecting NEK4 expression levels and post-translational modifications

• Immunohistochemistry (IHC) for visualizing NEK4 localization in tissue samples

• Immunofluorescence (IF) for subcellular localization studies

These applications are crucial for investigating NEK4's roles in senescence, DNA damage response, and cancer progression, allowing researchers to track its expression, localization, and functional interactions in various experimental contexts.

How do I optimize NEK4 antibody concentration for ELISA applications?

Optimizing NEK4 antibody concentration for ELISA requires systematic titration to achieve high specificity with minimal background. Based on manufacturer recommendations, start with a dilution range of 1:500-1:1000 for HRP-conjugated NEK4 antibodies . For optimal results:

• Perform a preliminary checkerboard titration using 2-fold serial dilutions of both antigen and antibody

• Include proper negative controls (e.g., isotype-matched IgG) to determine background signal

• Calculate signal-to-noise ratios for each concentration

• Select the dilution that provides maximum specific signal while maintaining low background

• Validate this concentration with known positive and negative samples

When working with recombinant NEK4 protein fragments (such as the 516-661AA region used as immunogen for some available antibodies), ensure that your target contains this epitope region for reliable detection .

How can NEK4 antibodies be used to investigate its role in DNA damage response pathways?

NEK4 antibodies can be leveraged to interrogate the protein's involvement in DNA damage response (DDR) through several sophisticated approaches:

• Co-immunoprecipitation assays: Use NEK4 antibodies to pull down protein complexes, followed by mass spectrometry or western blotting to detect known DDR proteins. Research has shown that NEK4 forms complexes with DNA-dependent protein kinase catalytic subunit [DNA-PK(cs)], Ku70, and Ku80 .

• Chromatin immunoprecipitation (ChIP): Apply NEK4 antibodies to investigate recruitment to damaged DNA sites.

• Proximity ligation assays: Combine NEK4 antibodies with antibodies against DDR components to visualize protein-protein interactions in situ.

• Immunofluorescence colocalization: Track NEK4 localization during DNA damage using dual-labeling with γH2AX or 53BP1 as DNA damage markers.

When NEK4 is suppressed, defects occur in the recruitment of DNA-PK(cs) to DNA following double-stranded breaks, resulting in reduced p53 activation and H2AX phosphorylation . These approaches can help elucidate NEK4's specific mechanistic contributions to DNA repair pathways.

What is the relationship between NEK4 and cancer metastasis, and how can antibodies help investigate this connection?

Recent studies have identified NEK4 as a novel kinase involved in regulating epithelial-mesenchymal transition (EMT), a critical process in cancer metastasis . NEK4 antibodies can help investigate this relationship through:

• Expression profiling: Compare NEK4 levels in primary tumors versus metastatic lesions using IHC or western blotting.

• Phospho-specific investigations: Develop or use phospho-specific antibodies to track NEK4 kinase activity during metastatic progression.

• Mechanistic pathway analysis: Combine NEK4 immunoprecipitation with analysis of EMT markers to elucidate signaling pathways.

Research demonstrates that NEK4 knockdown results in:

• Increased E-cadherin expression (an epithelial marker)

• Decreased ZEB1 expression (an EMT transcription factor)

• Reduced Smad3 activation (a TGF-β pathway transducer)

• Diminished cancer cell migration and invasion capabilities

In an in vivo pulmonary metastasis mouse model, NEK4 knockdown significantly reduced metastatic colony formation, with IHC showing a negative correlation between NEK4 and E-cadherin levels . These findings position NEK4 as a potential therapeutic target for preventing cancer metastasis.

How do the distinct isoforms of NEK4 differ functionally, and what antibody considerations apply?

NEK4 exists in at least two confirmed isoforms, Nek4.1 (isoform 1) and Nek4.2 (isoform 2), with distinct interaction profiles that suggest different cellular functions . When investigating these isoforms:

• Isoform specificity: Verify whether your NEK4 antibody recognizes both isoforms or is isoform-specific. Many commercial antibodies target the C-terminal region (e.g., AA 516-661), which may be present in both isoforms .

• Interactome differences: Nek4.1 has approximately three times more interaction partners (474) than Nek4.2 (150), with about 68% of Nek4.2 interactors (102 proteins) shared with Nek4.1 .

• Functional context: Experimental evidence shows unexpected differences between both isoforms in RNA splicing control, suggesting distinct regulatory roles .

When designing experiments to distinguish between isoform-specific functions:

• Use isoform-specific antibodies if available

• Employ isoform-specific knockdown or overexpression approaches

• Perform rescue experiments with each isoform separately

Understanding these isoform differences is crucial for accurate interpretation of NEK4-related data and may explain conflicting results observed across different experimental models.

What are the optimal conditions for preserving NEK4 antibody activity during experimental procedures?

To maintain optimal NEK4 antibody activity, especially HRP-conjugated variants:

• Storage conditions:

  • Store antibody at the recommended temperature (typically 2-8°C for short-term, -20°C for long-term)

  • Avoid repeated freeze-thaw cycles (aliquot upon receipt)

  • Protect from light, especially important for HRP-conjugated antibodies

• Working conditions:

  • Use freshly prepared buffers

  • Maintain pH stability (typically pH 7.2-7.6)

  • Add appropriate stabilizers when diluting (e.g., 1% BSA)

  • For HRP-conjugated antibodies, include 0.01% thimerosal or 0.05% ProClin 300 to prevent microbial growth

• Dilution protocols:

  • Prepare fresh working dilutions (1:500-1:1000 for ELISA applications)

  • Use high-quality diluents that maintain protein stability

  • Minimize exposure to strong oxidizing agents that can compromise HRP activity

These precautions are particularly important for preserving the dual functionality of NEK4 antibody-HRP conjugates, ensuring both specific binding to NEK4 epitopes and reliable enzymatic activity for detection.

What strategies can overcome non-specific binding issues with NEK4 antibodies?

Non-specific binding can significantly compromise NEK4 antibody performance. To address this challenge:

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, normal serum from host species)

  • Extend blocking time (2 hours to overnight) at 4°C

  • Include 0.1-0.3% Triton X-100 for enhanced antibody penetration in immunohistochemistry

• Antibody validation:

  • Confirm specificity using NEK4 knockout/knockdown samples

  • Perform peptide competition assays with the immunizing peptide (e.g., NEK4 AA 516-661)

  • Use multiple antibodies targeting different NEK4 epitopes to confirm results

• Sample preparation refinements:

  • Optimize fixation conditions (over-fixation can mask epitopes)

  • Include additional washing steps with increased salt concentration

  • For tissues, perform antigen retrieval optimization

• Dilution optimization:

  • Test broader dilution ranges than the recommended 1:500-1:1000

  • Reduce primary antibody concentration while extending incubation time

When troubleshooting, systematically alter one variable at a time while maintaining appropriate positive and negative controls to identify the source of non-specific binding.

How can I validate NEK4 antibody specificity in my experimental system?

Rigorous validation of NEK4 antibody specificity is essential for generating reliable data:

• Genetic approaches:

  • Test antibody reactivity in NEK4 knockout/knockdown systems (using shRNA or siRNA as described in publications)

  • Perform reciprocal validation with NEK4 overexpression systems

• Biochemical validation:

  • Conduct western blotting to confirm the antibody detects a band of the expected molecular weight (Nek4 is approximately 95 kDa)

  • Perform immunoprecipitation followed by mass spectrometry to confirm target identity

  • Use peptide competition assays with the immunizing peptide (NEK4 AA 516-661)

• Cross-reactivity assessment:

  • Test the antibody against other NEK family members (NEK1-11) to confirm specificity

  • Ensure antibody works in your specific species (current evidence supports human reactivity)

• Functional correlation:

  • Confirm that antibody-detected signals correlate with known NEK4 functions (e.g., DNA damage response activity, EMT regulation)

  • Use phospho-specific antibodies to correlate kinase activity with physiological outcomes

This multi-faceted validation approach ensures that observed signals genuinely represent NEK4 and not related proteins or artifacts.

How is NEK4 involved in cellular senescence, and what experimental approaches can investigate this connection?

NEK4 has been identified as a critical regulator of replicative senescence through RNA interference screening . To investigate this connection:

• Senescence phenotyping:

  • Monitor senescence-associated β-galactosidase activity in cells with manipulated NEK4 levels

  • Track population doubling levels (PDLs) in NEK4-suppressed versus control fibroblasts

  • Assess senescence markers (p21, p16) in relation to NEK4 expression

• Mechanistic investigations:

  • Examine NEK4's impact on p53-p21 pathway activation through western blotting

  • Analyze DNA damage foci formation using γH2AX staining

  • Assess telomere integrity in relation to NEK4 function

Research indicates that NEK4 suppression extends the number of population doublings required to reach replicative senescence in human fibroblasts and results in decreased transcription of p21, a key senescence mediator . Furthermore, NEK4-suppressed cells display impaired cell cycle arrest in response to double-stranded DNA damage, suggesting a mechanistic link between DNA damage sensing and senescence entry .

What is the significance of NEK4 in cancer research, and how are antibodies advancing this field?

NEK4 has emerged as a significant factor in cancer biology, particularly through its involvement in:

• Metastasis regulation: NEK4 promotes EMT in lung cancer, with NEK4 knockdown inhibiting migration, invasion, and metastasis formation in vivo .

• DNA damage response: NEK4 suppression causes defects in the recruitment of DNA-PK(cs) to damaged DNA, which has implications for cancer treatment sensitivity .

• Therapeutic targeting potential: Given its roles in metastasis and DNA repair, NEK4 represents a potential target for cancer therapy development.

NEK4 antibodies are advancing cancer research through:

• Biomarker development: IHC analysis of tumor samples to correlate NEK4 expression with clinical outcomes

• Mechanistic studies: Elucidating NEK4's interactions with EMT regulators (ZEB1, Smad3) and DNA repair machinery

• Therapeutic response prediction: Assessing NEK4 levels as potential predictors of response to DNA-damaging therapies

In lung cancer models, high NEK4 expression correlates with increased metastatic potential, and NEK4 knockdown significantly reduces pulmonary metastasis in vivo . These findings suggest that NEK4 antibodies could serve as valuable tools for patient stratification and treatment selection in precision oncology approaches.

How do NEK4 interaction partners influence its cellular functions, and what techniques can map this interactome?

NEK4 functions through a complex network of protein interactions that vary between its isoforms. To investigate this interactome:

• Immunoprecipitation-mass spectrometry (IP-MS): This approach has identified 474 interacting proteins for isoform 1 and 149 for isoform 2, with approximately 68% overlap .

• Proximity-dependent biotinylation (BioID): This can identify transient or weak interactions within the native cellular context.

• Yeast two-hybrid screening: Useful for identifying direct binary interactions with NEK4.

• Co-immunoprecipitation with specific antibodies: Confirms individual interactions identified through high-throughput methods.

Key NEK4 interaction partners with functional significance include:

These interaction studies reveal NEK4's involvement in diverse cellular processes beyond its known kinase function, positioning it as a multifunctional signaling hub.