NEK11 Antibody

What is NEK11 and why is it important in cell biology research?

NEK11 is a member of the NIMA (Never In Mitosis gene A) family of serine/threonine kinases that plays critical roles in DNA damage response and cell cycle regulation. It is particularly important for maintaining genomic integrity through its role in the G2/M checkpoint. Research shows that NEK11 is activated in response to genotoxic agents and controls the degradation of CDC25A by directly phosphorylating it on residues required for BTRC-mediated polyubiquitination and degradation .

NEK11's importance in research stems from its:

• Role in DNA damage checkpoint signaling

• Involvement in cancer biology, particularly colorectal and melanoma

• Function in genotoxic stress responses

• Participation in cell cycle regulation

What are the known isoforms of NEK11 and how do they differ functionally?

Four splice variants of NEK11 have been identified (L/S/C/D), each with distinct properties:

In HCT116 cells, NEK11S in particular has an important role in the DNA damage response . Research demonstrates that all variants undergo nucleocytoplasmic shuttling mediated through adjacent nuclear import and export signals in the C-terminal non-catalytic domain .

What applications are NEK11 antibodies commonly used for in research?

NEK11 antibodies are utilized across multiple research applications:

When selecting an antibody for specific applications, researchers should verify the validated applications for each antibody product and consider both monoclonal (e.g., OTI4H5, 36JK) and polyclonal options depending on experimental needs .

How should I design experiments to study NEK11's role in DNA damage response pathways?

When designing experiments to investigate NEK11's function in DNA damage response, consider this methodological framework:

• Cell Model Selection:

  • Use established cell lines like HCT116 (with and without p53) to compare p53-dependent effects

  • Consider using cells from relevant cancer types (colorectal cancer or melanoma cells)

• Manipulation Approaches:

  • RNAi-mediated depletion using validated siRNAs targeting NEK11

  • Overexpression of different NEK11 isoforms (especially NEK11S for DNA damage studies)

  • CRISPR/Cas9 knockout models

• Damage Induction:

  • Ionizing radiation (10 Gy effective for G2/M arrest in HCT116 cells)

  • Topoisomerase I inhibitors (irinotecan at 5 μM for 20 hours)

• Analytical Methods:

  • Flow cytometry with PI staining for cell cycle analysis (collect cells 16 hours post-treatment)

  • Western blotting to confirm knockdown and analyze target proteins

  • Apoptosis assays (Annexin V/PI staining)

  • Clonogenic survival assays (use 2 Gy for long-term studies)

  • Immunofluorescence to track subcellular localization

Research demonstrates that NEK11 depletion prevents G2/M arrest induced by genotoxic agents and promotes p53-dependent apoptosis both in the presence and absence of DNA damage .

What controls should be included when validating NEK11 antibody specificity?

Proper validation of NEK11 antibody specificity requires multiple controls:

• Positive Controls:

  • Cells with known NEK11 expression (HeLa cells have been verified)

  • Recombinant NEK11 protein or NEK11-overexpressing cells (HEK293T transfected with NEK11)

  • Testing across tissues with known expression patterns

• Negative Controls:

  • NEK11 knockdown or knockout cells

  • Secondary antibody-only controls

  • Non-specific IgG controls of the same isotype

  • Blocking peptide competition assays

• Specificity Tests:

  • Western blot to confirm the correct molecular weight (~54-74 kDa depending on isoform)

  • Testing reactivity across species if performing comparative studies

  • Cross-reactivity assessment with other NEK family members (especially close paralogs)

  • Testing multiple antibodies targeting different NEK11 epitopes

• Application-Specific Controls:

  • For IF/IHC: Include subcellular markers to confirm expected localization patterns

  • For WB: Include loading controls and molecular weight markers

Researchers have reported that NEK11 detection can be challenging due to its low endogenous expression in many cell types, necessitating careful optimization of detection methods .

How should NEK11 antibodies be optimized for immunofluorescence studies of subcellular localization?

Optimizing NEK11 antibodies for immunofluorescence requires careful attention to several methodological details:

• Fixation Method Selection:

  • Test both paraformaldehyde (PFA) (4%, 10-15 minutes) and methanol fixation (-20°C, 10 minutes)

  • NEK11's nuclear/nucleolar localization may be better preserved with certain fixatives

• Permeabilization Optimization:

  • Test different permeabilization agents (0.1-0.5% Triton X-100, 0.1-0.5% Saponin)

  • Determine optimal permeabilization time (5-15 minutes)

• Blocking Conditions:

  • Use 5-10% normal goat serum (NGS) in PBS/Tween

  • Consider adding BSA (1-3%) to reduce background

• Antibody Dilution Optimization:

  • Start with manufacturer's recommendation (typically 1:100-1:1000)

  • Perform dilution series to determine optimal signal-to-noise ratio

  • Incubate primary antibody overnight at 4°C

• Validation with Known Localization Patterns:

  • NEK11 is primarily cytoplasmic but shuttles to the nucleus

  • Use leptomycin B (LMB), a nuclear export inhibitor, to validate nuclear accumulation (all four NEK11 variants accumulate in the nucleus after LMB treatment)

  • Co-stain with nucleolar markers to verify nucleolar localization

• Advanced Imaging Considerations:

  • Use confocal microscopy for detailed subcellular localization

  • Consider super-resolution techniques for studying subnuclear structures

  • Perform co-localization studies with cell cycle markers

Research demonstrates that while NEK11L and NEK11D isoforms are restricted to the cytoplasm, NEK11S and NEK11C are detected in both the cytoplasm and nucleus, highlighting the importance of isoform-specific analysis .

How can NEK11 antibodies be utilized to study cell cycle-dependent localization patterns?

Studying NEK11's dynamic localization through the cell cycle requires sophisticated approaches:

• Cell Synchronization Protocols:

  • Double thymidine block for G1/S boundary

  • Nocodazole treatment for prometaphase

  • Serum starvation for G0

• Cell Cycle Stage Verification:

  • Co-stain with stage-specific markers (cyclin B1, pH3, PCNA)

  • Perform parallel flow cytometry to verify synchronization efficiency

• Advanced Imaging Approaches:

  • Live cell imaging with fluorescently tagged NEK11 constructs

  • Fixed cell analysis at defined time points post-synchronization release

  • 3D confocal imaging with z-stack acquisition

• Quantitative Analysis Methods:

  • Nuclear/cytoplasmic signal ratio quantification

  • Colocalization analysis with organelle markers

  • Tracking of NEK11 during mitotic progression

Research shows that NEK11 accumulates in the nucleolus in G1/S-arrested cells and localizes to polar microtubules during prometaphase and metaphase . The nuclear-cytoplasmic distribution is regulated by sequences in the C-terminal domain - the coiled-coil regions (residues 1-388) contain sequences necessary for nuclear import, while the region between residues 388 and 465 contains sequences necessary for nuclear export .

What approaches can resolve contradictory findings regarding NEK11's role in the DNA damage checkpoint?

Resolving contradictions in NEK11 function requires systematic investigation:

• Isoform-Specific Analysis:

  • Develop isoform-specific detection methods (antibodies or tagged constructs)

  • Perform selective knockdown/knockout of individual isoforms

  • Express individual isoforms in knockout backgrounds

• Context-Dependent Studies:

  • Analyze NEK11 function across multiple cell types

  • Compare function in normal versus cancer cells

  • Evaluate different DNA damaging agents (IR vs. irinotecan)

• Interaction Network Analysis:

  • Perform co-immunoprecipitation with candidate interactors

  • Conduct proximity labeling experiments (BioID, APEX)

  • Map phosphorylation networks downstream of NEK11

• Kinase Activity Assessment:

  • Develop in vitro kinase assays for NEK11

  • Compare wild-type vs. kinase-dead mutants

  • Identify substrate specificities for different isoforms

While some studies suggest NEK11 directly phosphorylates CDC25A on sites within a phosphodegron that promotes β-TrCP recruitment , others argue that phosphorylation-dependent degradation of CDC25 may be mediated by alternative kinases like casein kinase 1 . A comprehensive analysis of substrate specificity may help resolve these contradictions. Notably, one study reported that while all NEK kinases were systematically studied for phosphorylation-site motifs, NEK11 could not be successfully analyzed due to insufficient activity, suggesting technical challenges in studying this kinase .

How can NEK11 antibodies be applied to understand its potential role in cancer progression?

Investigating NEK11's role in cancer requires multiple methodological approaches:

• Expression Analysis in Clinical Samples:

  • IHC on tissue microarrays across cancer stages

  • Correlation with patient outcomes and tumor features

  • Comparison between tumor and matched normal tissue

• Functional Studies in Cancer Models:

  • NEK11 manipulation (knockdown/overexpression) in cancer cell lines

  • Assessment of proliferation, apoptosis, and DNA damage response

  • Xenograft studies with NEK11-modified cells

• Therapy Response Correlation:

  • Analyze NEK11 levels before and after chemotherapy/radiotherapy

  • Correlate NEK11 expression with treatment response

  • Test combination of NEK11 inhibition with standard therapies

• Genetic Analysis:

  • Screen for NEK11 mutations in cancer cohorts

  • Investigate the functional consequences of cancer-associated variants

  • Analyze loss of heterozygosity at the NEK11 locus

Research indicates that NEK11 expression is upregulated in early-stage colorectal cancers (CRCs) , and a rare nonsense variant in the NEK11 gene (c.1120C>T, p.Arg374Ter) has been identified as a potential high-penetrance melanoma susceptibility mutation . In melanoma from a variant carrier, somatic loss of the wildtype allele of this putative tumor suppressor gene was demonstrated, and functional analyses showed that the NEK11 p.Arg374Ter mutation results in strongly reduced expression of the truncated protein caused by proteasomal degradation .

How should researchers address challenges in detecting endogenous NEK11 expression?

Detecting endogenous NEK11 presents several challenges that can be addressed through these approaches:

• Sample Preparation Optimization:

  • Enrich nuclear fractions when studying nuclear NEK11

  • Use phosphatase inhibitors to preserve phosphorylated forms

  • Consider proteasome inhibitors (MG132) to prevent degradation of unstable isoforms like NEK11D

• Signal Amplification Techniques:

  • Use high-sensitivity detection systems for Western blot

  • Consider tyramide signal amplification for IHC/IF

  • Employ sandwich ELISA approaches for quantitative detection

• Enrichment Strategies:

  • Perform immunoprecipitation before Western blotting

  • Consider using inducible expression systems as positive controls

  • Induce DNA damage to upregulate NEK11 expression/activity

• Alternative Detection Methods:

  • RT-PCR to assess transcript levels

  • RNA-FISH to visualize transcript localization

  • Mass spectrometry for protein identification

Research shows that NEK11 expression and activity are elevated in cells exposed to DNA damaging agents and replication inhibitors , suggesting these treatments may help increase detection sensitivity in experimental systems.

What strategies can overcome limitations in studying NEK11 kinase activity in research applications?

Studying NEK11 kinase activity presents unique challenges that can be addressed through specialized techniques:

• Activity-Based Assay Design:

  • Develop phospho-specific antibodies for NEK11 substrates

  • Use in vitro kinase assays with recombinant substrates

  • Employ ATP analog-sensitive mutants for specific activity tracking

• Activation Strategies:

  • Induce DNA damage via ionizing radiation (10 Gy)

  • Treat with topoisomerase inhibitors like irinotecan (5 μM)

  • Consider Chk1 activation to drive NEK11 phosphorylation on Ser-273

• Protein Engineering Approaches:

  • Generate constitutively active NEK11 mutants

  • Create kinase-dead versions as negative controls

  • Design FRET-based reporters for NEK11 activity

• Advanced Mass Spectrometry:

  • Phosphoproteomics to identify NEK11 substrates

  • Chemical proteomics with modified ATP analogs

  • Quantitative assessment of phosphorylation stoichiometry

How can researchers distinguish between NEK11 and other NEK family members in their experiments?

Differentiating NEK11 from other NEK family members requires careful experimental design:

• Antibody Selection Strategies:

  • Choose antibodies raised against unique regions (non-conserved C-terminal domains)

  • Validate specificity against recombinant NEK family proteins

  • Consider using isoform-specific antibodies when available

• Expression Analysis Approaches:

  • Design PCR primers targeting unique exons

  • Use RNA sequencing to distinguish transcript isoforms

  • Perform careful Western blot analysis to distinguish by molecular weight

• Functional Differentiation:

  • Compare phenotypes of specific NEK knockdowns

  • Analyze subcellular localization patterns

  • Examine response to specific cellular stresses

• Advanced Validation Methods:

  • Perform rescue experiments with specific NEK family members

  • Use CRISPR/Cas9 tagging at endogenous loci

  • Conduct comprehensive immunoprecipitation specificity tests

The NEK family consists of eleven members (NEK1-11), several of which have roles in the DNA damage response. Research shows that at least four (NEK1, NEK8, NEK10, and NEK11) have suspected roles in the DNA damage response . Distinguishing NEK11's specific functions from these related kinases requires careful experimental controls and validation approaches.

How can NEK11 antibodies be applied to study its potential role in mitochondrial function?

While NEK11's direct role in mitochondrial function is not yet well-established, research on other NEK family members suggests potential approaches:

• Co-localization Studies:

  • Perform immunofluorescence with mitochondrial markers

  • Conduct subcellular fractionation and Western blotting

  • Use proximity ligation assays to detect potential mitochondrial interactions

• Functional Mitochondrial Assays:

  • Measure mitochondrial respiration in NEK11-depleted cells

  • Assess mitochondrial membrane potential

  • Analyze ROS production and mitochondrial stress responses

• Protein Interaction Analysis:

  • Identify potential mitochondrial binding partners

  • Screen for interactions with mitochondrial import machinery

  • Investigate possible roles in mitochondrial dynamics

• Genetic Approaches:

  • Compare mitochondrial phenotypes in NEK11 knockout models

  • Analyze mitochondrial DNA integrity

  • Evaluate expression of mitochondrial genes

Recent research has demonstrated that several NEK family members (NEK1, NEK4, NEK5, NEK6, and NEK10) have roles in controlling mitochondrial functions including respiration, dynamics, mtDNA maintenance, and stress response . While NEK11's mitochondrial function is not yet established, the family connection suggests potential research directions worth exploring.

What are the best approaches to study NEK11 in patient-derived cancer models?

Investigating NEK11 in patient-derived cancer models requires specialized techniques:

• Patient-Derived Xenograft (PDX) Models:

  • Immunohistochemical analysis of NEK11 expression

  • Correlation with tumor growth and therapy response

  • Assessment of NEK11 phosphorylation status

• Patient-Derived Organoids:

  • Establish colorectal cancer or melanoma organoids

  • Manipulate NEK11 expression via lentiviral approaches

  • Test sensitivity to DNA-damaging therapies

• Clinical Sample Analysis:

  • Develop tissue microarrays for high-throughput analysis

  • Perform multiplex immunofluorescence to assess NEK11 in tumor microenvironment

  • Correlate NEK11 levels with clinical parameters and outcomes

• Genetic Screening:

  • Screen for NEK11 mutations in cancer patient cohorts

  • Generate knock-in models of patient-derived mutations

  • Assess functional consequences of cancer-associated variants

Research has identified NEK11 as a potentially relevant cancer biomarker, with elevated expression detected in colorectal adenomas and a nonsense variant in the NEK11 gene (c.1120C>T, p.Arg374Ter) identified in a Dutch family with melanoma and characterized as a potential novel high-penetrance melanoma-susceptibility gene .

How can advanced imaging techniques enhance our understanding of NEK11 dynamics during DNA damage response?

Advanced imaging approaches offer unique insights into NEK11 function:

• Live Cell Imaging Strategies:

  • Generate fluorescently tagged NEK11 constructs for live imaging

  • Employ photoactivatable or photoconvertible tags for pulse-chase analysis

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to measure mobility

• Super-Resolution Microscopy Applications:

  • Use STED or STORM microscopy for nanoscale localization

  • Analyze NEK11 clustering at DNA damage sites

  • Determine precise nuclear subdomains containing NEK11

• Multi-dimensional Imaging:

  • Combine time-lapse with z-stack acquisition

  • Perform spectral imaging with multiple markers

  • Implement correlative light and electron microscopy

• Quantitative Image Analysis:

  • Develop algorithms for tracking NEK11 dynamics

  • Measure recruitment kinetics to DNA damage sites

  • Quantify co-localization with DNA repair factors

Research shows that NEK11 undergoes dynamic relocalization during cell cycle progression and in response to DNA damage, suggesting that advanced imaging approaches could reveal important functional details about its recruitment and activity at sites of DNA damage .