NUCKS1 Antibody

What epitopes should researchers target when selecting NUCKS1 antibodies?

NUCKS1 contains several key structural domains that could serve as potential epitope targets. The most critical regions include the DNA-binding domain (which includes an AT-hook motif), the two nuclear localization sequences (NLS1 and NLS2), and the extensively modified regions containing phosphorylation sites. When selecting antibodies, researchers should consider whether their experimental questions require detection of specific post-translational modifications, as NUCKS1 is one of the most heavily modified proteins in the human proteome . For detection of total NUCKS1 regardless of modification state, antibodies targeting the conserved regions outside major phosphorylation sites are preferable. For studying specific functions, such as DNA binding activity, antibodies specifically recognizing the DNA-binding domain (with preference for GC-rich sequences) should be considered .

How does NUCKS1 antibody specificity differ between various experimental applications?

The required antibody specificity varies significantly depending on the experimental approach. For immunohistochemistry applications, antibodies need to maintain specificity under fixation conditions that may alter protein conformation. Evidence suggests NUCKS1 serves as a novel immunohistochemical marker for early detection of hepatocellular carcinoma, requiring antibodies with high specificity in fixed tissues . For ChIP-qPCR experiments mapping NUCKS1 binding to promoters (like the SKP2 promoter), antibodies must efficiently precipitate NUCKS1-DNA complexes without cross-reactivity to the highly homologous RAD51AP1 protein . In Western blot applications, antibodies must distinguish between phosphorylated and non-phosphorylated forms, as dephosphorylation significantly increases NUCKS1's DNA-binding affinity, changing its functional properties nearly 10-fold .

What validation methods are essential for confirming NUCKS1 antibody specificity?

Rigorous validation of NUCKS1 antibodies is critical due to its sequence homology with RAD51AP1 and extensive post-translational modifications. At minimum, validation should include:

• Western blot comparison between wild-type cells and NUCKS1 knockout/knockdown cells (e.g., using CRISPR/Cas9 or siRNA techniques as described in the literature)

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Immunofluorescence comparison in cells with and without NUCKS1 expression

• Peptide competition assays to verify epitope specificity

• Testing across multiple cell lines, as NUCKS1 expression varies significantly between tissues and cancer types

Cross-reactivity testing against RAD51AP1 is particularly important since these proteins share extensive sequence homology throughout their structure . For phospho-specific antibodies, validation should include treatment with lambda phosphatase to confirm specificity for the phosphorylated form .

How should researchers optimize chromatin immunoprecipitation (ChIP) protocols when using NUCKS1 antibodies?

Optimizing ChIP protocols for NUCKS1 requires special consideration of its chromatin-association properties and DNA-binding preferences. Based on published research:

• Crosslinking conditions: NUCKS1 interacts with GC-rich DNA sequences, particularly at promoter regions like the SKP2 promoter . Standard formaldehyde fixation (1%) for 10 minutes is typically sufficient, but dual crosslinking with both formaldehyde and protein-specific crosslinkers may improve efficiency.

• Sonication parameters: Aim for chromatin fragments between 200-500bp to effectively capture NUCKS1 binding sites, which often correspond to promoter regions directly upstream of transcription start sites.

• Antibody selection: Use antibodies validated specifically for ChIP applications. For mapping binding sites (as shown in SKP2 promoter studies), design qPCR primers covering sequential regions of target promoters with particular attention to GC-rich regions .

• Controls: Include both input controls and ChIP with IgG. Additionally, validation in NUCKS1-depleted cells is essential to confirm signal specificity.

• Cell cycle considerations: NUCKS1 recruitment to chromatin is stimulated by mitogens and varies throughout the cell cycle, peaking in late G1 phase . Synchronizing cells or documenting cell cycle stage distribution is recommended for reproducible results.

What are the key considerations when designing immunofluorescence experiments to study NUCKS1 localization patterns?

When designing immunofluorescence experiments for NUCKS1:

• Fixation method: Standard paraformaldehyde (4%) fixation preserves NUCKS1 nuclear localization, but methanol fixation may better preserve nuclear architecture for co-localization studies with DNA repair factors.

• Cell cycle stage: NUCKS1 shows cell cycle-dependent localization and activity patterns. It is crucial for S phase entry and exhibits different binding patterns throughout the cell cycle . Consider using cell cycle markers (e.g., EdU for S-phase) for co-staining.

• DNA damage induction: Upon exposure to DNA-damaging agents, NUCKS1 controls the resolution of RAD54 foci . Time-course experiments following damage induction (IR, MMC, or other agents) are valuable for studying its dynamic relocalization.

• Co-localization: Design co-staining with key interaction partners such as RAD54, RAD51, or components of the SKP2-p21/p27 axis .

• Signal specificity: Include appropriate controls using NUCKS1-depleted cells to confirm antibody specificity, as nuclear staining can often show background signals.

• Resolution requirements: Since NUCKS1 forms discrete nuclear foci after DNA damage, super-resolution microscopy may be necessary to fully resolve its spatial relationship with other DNA repair proteins.

How can researchers effectively use NUCKS1 antibodies to study its role in DNA damage response?

NUCKS1 plays critical roles in homologous recombination DNA repair and functions as a tumor suppressor in mice . To effectively study these aspects:

• Time-course experiments: Following DNA damage induction (particularly with agents causing double-strand breaks), monitor NUCKS1 dynamics at different timepoints. NUCKS1 is required for the timely resolution of DNA damage-induced RAD54 foci .

• Co-immunoprecipitation: Use NUCKS1 antibodies for co-IP experiments to capture its interactions with repair proteins like RAD54. This approach can reveal how NUCKS1 prevents RAD54's inappropriate engagement with RAD51AP1 in unperturbed cells .

• Phosphorylation status: Use phospho-specific antibodies to monitor ATM-dependent phosphorylation of NUCKS1 at Ser54 after DNA damage. This modification is important for its function in the DNA damage response .

• Functional assays: Combine antibody detection with functional assays such as HR reporter assays after NUCKS1 depletion to correlate protein levels with repair efficiency.

• Cancer cell lines: Compare NUCKS1 levels and localization patterns across different cancer cell lines, as NUCKS1 expression is altered in various cancers and may correlate with therapy resistance .

How can researchers utilize NUCKS1 antibodies to investigate its dual role in both tumor suppression and oncogenesis?

NUCKS1 exhibits a complex, context-dependent role in cancer biology, functioning as both a tumor suppressor and potential oncogenic factor depending on the cancer type and cellular context . To investigate this duality:

• Tissue microarray analysis: Use validated NUCKS1 antibodies on tissue microarrays containing multiple cancer types to quantitatively assess expression patterns. NUCKS1 is elevated in hepatocellular carcinoma, lung cancer, and several other malignancies, but decreased in adult T-cell leukemia-lymphoma and childhood acute lymphoblastic leukemia .

• Correlation studies: Combine NUCKS1 immunostaining with markers of DNA damage (γH2AX), cell proliferation (Ki-67), and known tumor suppressors (p53) to identify patterns. NUCKS1 is inversely correlated with miR-137 expression in lung cancer .

• Functional genomics approach: In knockdown/overexpression models, use antibodies to confirm protein levels while measuring oncogenic phenotypes (proliferation, invasion, therapy resistance).

• Post-translational modification analysis: Use modification-specific antibodies to determine how phosphorylation patterns of NUCKS1 differ between normal and cancer cells, as these modifications significantly affect its function .

• Mouse models: For in vivo studies, analyze NUCKS1 expression in Trp53+/− NUCKS1+/− mice, which develop tumors more rapidly than Trp53+/− mice after irradiation, indicating its tumor suppressor function .

What approaches can researchers take to study NUCKS1's role in metabolic regulation using antibodies?

NUCKS1 has been implicated in metabolic regulation with important implications for obesity and insulin resistance . Researchers can:

• Tissue-specific analysis: Use NUCKS1 antibodies to compare protein levels in adipose tissue between lean and obese subjects. Studies show >40% reduction in NUCKS1 protein in overweight individuals compared to lean individuals .

• Hypothalamic expression: Investigate NUCKS1 levels in hypothalamic sections, as hypothalamus-specific deletion of NUCKS1 (HNKO) leads to increased body weight/fat mass and impaired glucose tolerance .

• Insulin signaling pathway: Combine NUCKS1 staining with phospho-specific antibodies for insulin signaling components to correlate NUCKS1 levels with pathway activation.

• Nutritional interventions: Monitor NUCKS1 protein levels in response to various dietary interventions or fasting/feeding cycles.

• Relationship with p53: Investigate the inverse relationship between p53 and NUCKS1 in adipose tissue, as upregulated p53 has been linked to insulin resistance and can downregulate NUCKS1 .

• Chromatin studies: Use ChIP-seq with NUCKS1 antibodies to identify metabolic genes directly regulated by NUCKS1 in relevant tissues.

How can phospho-specific NUCKS1 antibodies be utilized to dissect its differential functions?

NUCKS1 is extensively post-translationally modified, and its phosphorylation status dramatically affects its function and binding properties . To leverage phospho-specific antibodies:

• Phospho-site mapping: Use phospho-specific antibodies targeting key sites (such as ATM-dependent phosphorylation at Ser54) to track modification status after DNA damage .

• Functional correlation: Compare DNA binding activity of NUCKS1 with its phosphorylation state. Dephosphorylated NUCKS1 shows nearly 10-fold higher affinity for the SKP2 promoter in EMSA assays .

• Kinase inhibition studies: Combine specific kinase inhibitors (CDK, casein kinase, ATM) with phospho-specific antibody detection to identify regulatory pathways controlling NUCKS1 activity.

• Cell cycle analysis: Track changes in phosphorylation patterns throughout the cell cycle, particularly during G1/S transition where NUCKS1 regulates SKP2 expression .

• Mutational analysis: Compare antibody reactivity between wild-type NUCKS1 and phospho-mutant versions to validate specificity and understand functional consequences.

What are the common pitfalls when detecting NUCKS1 in Western blots and how can they be addressed?

Common challenges in NUCKS1 Western blot detection include:

• Multiple bands: NUCKS1 undergoes extensive post-translational modifications, resulting in multiple bands that may be misinterpreted as non-specific binding. Resolution can be improved by:

  • Using gradient gels (7-15%) to better separate modified forms

  • Including phosphatase-treated controls to identify which bands represent phosphorylated species

  • Using longer running times for better separation of closely migrating forms

• Cross-reactivity with RAD51AP1: Due to sequence homology, antibodies may cross-react with RAD51AP1. To address this:

  • Use NUCKS1 knockout/knockdown controls

  • Pre-absorb antibodies with purified RAD51AP1 protein

  • Select antibodies targeting regions with minimal homology between the two proteins

• Molecular weight discrepancies: Despite its calculated molecular weight of 27 kDa, NUCKS1 often migrates at approximately 43 kDa in SDS-PAGE due to its unusual amino acid composition and post-translational modifications . Researchers should:

  • Include purified recombinant NUCKS1 as a size reference

  • Use size markers that cover the 25-50 kDa range

  • Consider using phospho-tag gels to better separate modified forms

• Low signal: NUCKS1 expression can be significantly reduced in certain tissues or disease states . To enhance detection:

  • Use more sensitive detection methods (e.g., chemiluminescence or fluorescent secondaries)

  • Enrich NUCKS1 by immunoprecipitation before Western blotting

  • Optimize transfer conditions for proteins in the 25-50 kDa range

How can researchers distinguish between genuine NUCKS1 signal and artifacts in immunohistochemistry?

Distinguishing genuine NUCKS1 staining from artifacts in immunohistochemistry requires:

• Comprehensive controls:

  • Tissue from NUCKS1 knockout models or validated knockdown samples

  • Comparison with in situ hybridization for NUCKS1 mRNA

  • Competitive blocking with immunizing peptide

  • Secondary-only controls to rule out non-specific binding

• Expected localization patterns: NUCKS1 is predominantly nuclear in most cell types, with some tissue-specific variations. Diffuse cytoplasmic staining is likely non-specific .

• Developmental context: NUCKS1 expression is elevated during early embryonic development and then gradually decreases until birth in most tissues except for nervous tissue and muscle fibers . This developmental pattern can serve as an internal validation.

• Tissue-specific benchmarks: NUCKS1 is highly expressed in stem cells and brain tissue but shows varied expression in other tissues . Compare staining intensity with established patterns.

• Dual staining approaches: Co-stain with antibodies against known NUCKS1 interactors (RAD54, components of the SKP2 pathway) to confirm biologically relevant localization .

• Antibody dilution series: Perform staining with a dilution series to identify the optimal antibody concentration that maximizes specific signal while minimizing background.

What strategies can address variability in NUCKS1 antibody performance across different experimental batches?

Batch-to-batch variability is a significant challenge in antibody-based research. For NUCKS1 antibodies, strategies include:

• Internal standardization:

  • Maintain a reference batch of validated antibody to benchmark new lots

  • Create standardized lysates from cell lines with consistent NUCKS1 expression for quality control

  • Document lot-specific optimal dilutions and incubation conditions

• Recombinant NUCKS1 standards:

  • Use purified recombinant NUCKS1 (as described for EMSA experiments) as a standard for calibration

  • Include gradient amounts of standard protein in Western blots to create standard curves

• Pooling strategy:

  • For critical experiments, consider pooling multiple antibody lots after individual validation

  • This reduces impact of lot-to-lot variability while maintaining sensitivity

• Application-specific validation:

  • Validate each new antibody lot specifically for intended applications (Western blot, IP, ChIP, IHC)

  • Different applications may have different sensitivities to lot variations

• Monoclonal alternatives:

  • Consider switching to monoclonal antibodies for reduced batch variation

  • Clone-specific validation can improve consistency

• Epitope mapping:

  • Characterize the exact epitope recognized by each antibody lot

  • Antibody lots recognizing the same epitope will likely perform more consistently

How can NUCKS1 antibodies be used to investigate its role in inflammatory and immune responses?

NUCKS1 has emerging roles in inflammatory signaling and immune responses that warrant investigation :

• NF-κB pathway interaction: NUCKS1 regulates NF-κB activation and the expression of specific NF-κB-mediated cytokines. Researchers can use NUCKS1 antibodies in combination with phospho-IκB antibodies to study this regulation in different cell types .

• Cytokine expression analysis: NUCKS1 knockout corneal epithelial cells show reduced expression of inflammatory mediators including IL6, IP10, and TNFα after LPS stimulation. Combine NUCKS1 staining with cytokine detection to map these relationships in different inflammatory contexts .

• HIV research applications: NUCKS1 is highly expressed in HIV-positive patients and increases Tat-mediated transcriptional activity of the HIV-1 LTR in an NF-κB-independent manner. Antibodies can help track NUCKS1 dynamics during viral infection .

• Tissue-specific inflammation: Since suppressed expression of inflammatory cytokines was observed in alkali-burned corneas of NUCKS1-deficient models, antibodies can help track NUCKS1's role in tissue-specific inflammatory responses .

• Co-immunoprecipitation studies: Use NUCKS1 antibodies to identify novel interaction partners in immune cells under different activation states.

• Chromatin dynamics: Perform ChIP-seq with NUCKS1 antibodies in immune cells before and after stimulation to identify inflammation-responsive target genes.

What methodological approaches can be used to study NUCKS1's interactions with the RAD54-RAD51 complex in DNA repair?

NUCKS1 physically and functionally interacts with the DNA motor protein RAD54 and influences the RAD51-mediated strand invasion step during homologous recombination . To study these interactions:

• Proximity ligation assays: Use NUCKS1 and RAD54/RAD51 antibodies in combination for in situ detection of protein-protein interactions at DNA damage sites.

• Sequential ChIP (re-ChIP): Perform ChIP with RAD54 antibodies followed by re-ChIP with NUCKS1 antibodies to identify genomic loci where both proteins co-localize.

• Immunofluorescence time-course: Track the temporal dynamics of NUCKS1, RAD54, and RAD51 localization after DNA damage using specific antibodies. NUCKS1 is required for the timely resolution of DNA damage-induced RAD54 foci .

• In vitro reconstitution: Combine purified components with antibody-based detection methods to study how NUCKS1 stimulates the ATPase activity of RAD54 and RAD51-RAD54-mediated strand invasion.

• Competitive binding assays: Use antibodies to detect how NUCKS1 prevents RAD54's inappropriate engagement with RAD51AP1 in unperturbed cells .

• Domain-specific antibodies: Employ antibodies recognizing specific domains of NUCKS1 to determine which regions mediate interactions with RAD54 and influence its ATPase activity.

How can researchers leverage NUCKS1 antibodies to explore its potential as a biomarker in various diseases?

NUCKS1's differential expression across various pathological conditions suggests potential biomarker applications :

• Cancer diagnostic development:

  • NUCKS1 is a novel immunohistochemical marker for early detection of hepatocellular carcinoma

  • Elevated in human lung cancer tissues and inversely correlated with miR-137 expression levels

  • Develop tissue microarray screening approaches with standardized antibody protocols to assess diagnostic value across cancer types

• Metabolic syndrome assessment:

  • NUCKS1 protein levels show inverse correlation with body mass index and insulin resistance markers

  • Establish standardized ELISA or immunoassay protocols for adipose tissue biopsies to correlate NUCKS1 levels with metabolic parameters

• Neurodegenerative disease research:

  • Given NUCKS1's association with Parkinson's disease and high expression in brain tissue , develop immunohistochemical protocols for neural tissues

  • Investigate cerebrospinal fluid detection methods to assess potential as a biofluid marker

• Multiparameter analysis:

  • Combine NUCKS1 antibodies with other established biomarkers in multiplexed assays

  • Correlate phosphorylation status with disease progression for more specific detection

• Therapy response prediction:

  • Monitor NUCKS1 levels before and after treatment (particularly DNA-damaging therapies)

  • Since NUCKS1 knockdown sensitizes cells to DNA-damaging agents like mitomycin C , antibody-based detection might help predict therapy response

• Longitudinal studies:

  • Develop standardized protocols for preserving and detecting NUCKS1 in archived samples

  • Enable retrospective studies correlating NUCKS1 levels with disease outcomes