Phospho-NUDC (Ser326) Antibody

What is NUDC protein and what is the significance of its phosphorylation at Ser326?

NUDC (Nuclear migration protein nudC) is a crucial protein that plays multiple essential roles in cellular processes including neurogenesis, neuronal migration, mitosis and cytokinesis. The protein is involved in spindle formation during mitosis and in microtubule organization during cytokinesis .

Phosphorylation at Ser326 is particularly significant as it occurs during the M phase of the cell cycle and is necessary for the correct formation of mitotic spindles and chromosome separation during mitosis. This post-translational modification is mediated by Polo-like kinase (PLK) and other kinases . The reversible phosphorylation of this residue serves as a regulatory mechanism for NUDC's function in cell division processes.

What are the main applications for Phospho-NUDC (Ser326) Antibody?

Phospho-NUDC (Ser326) Antibody can be utilized in multiple research applications:

• Western Blot (WB): Typically at dilutions of 1:500-1:2000

• Immunohistochemistry (IHC): Usually at dilutions of 1:100-1:300

• Immunofluorescence (IF): Recommended at dilutions of 1:50-200

• ELISA: Often at dilutions around 1:5000

• Cell-Based Phosphorylation ELISA: For analyzing phosphorylation in adherent cell cultures

These techniques enable researchers to detect endogenous levels of NUDC protein specifically when phosphorylated at S326, making it valuable for studying cell cycle regulation and mitotic processes.

What species reactivity does Phospho-NUDC (Ser326) Antibody exhibit?

Most commercially available Phospho-NUDC (Ser326) antibodies demonstrate cross-reactivity with human, mouse, and rat samples . This cross-species reactivity makes the antibody particularly valuable for comparative studies and allows researchers to investigate conserved phosphorylation-dependent mechanisms across these mammalian species.

How should I validate the specificity of Phospho-NUDC (Ser326) Antibody?

Validation of phospho-specific antibodies requires several critical steps:

• Lambda Phosphatase Treatment: Treat your samples with lambda phosphatase and compare with untreated samples. The antibody should not react with dephosphorylated protein, confirming its phospho-specificity .

  Protocol:

  • Incubate your protein sample with lambda phosphatase (200 ng) in reaction buffer (50 mM Tris-HCl, 100 mM NaCl, 2 mM MnCl₂, 2 mM DTT, 0.1 mM EGTA, 0.01% Brij 35, pH 7.5) for 1 hour at 30°C

  • Terminate reaction by adding SDS-PAGE loading buffer and heating at 100°C for 5 minutes

  • Run SDS-PAGE and Western blot with the phospho-antibody

• Peptide Competition Assay: Pre-incubate the antibody with the phosphorylated peptide before application to your samples. This should block antibody binding and eliminate signal .

• Western Blot Comparison: Compare reactivity with samples known to contain phosphorylated versus non-phosphorylated forms of the protein .

• Testing Multiple Cell/Tissue Types: Validate across different sample types where phosphorylation states are known to differ .

What controls should I include in experiments using Phospho-NUDC (Ser326) Antibody?

For rigorous experimental design, include these controls:

• Positive Control: Samples from M-phase cells where NUDC is known to be phosphorylated at Ser326 (e.g., nocodazole-arrested cells)

• Negative Controls:

  • Lambda phosphatase-treated samples

  • G1-phase synchronized cells (lower phosphorylation expected)

  • Non-specific IgG control of the same isotype and host species

• Peptide Blocking Control: Pre-incubate antibody with phospho-peptide immunogen to demonstrate binding specificity

• Loading Controls: For Western blots, include total NUDC antibody on stripped membranes to normalize phospho-signal to total protein levels

• Cross-reactivity Controls: Test the antibody against related phospho-proteins to ensure specificity

What are the optimal storage and handling conditions for Phospho-NUDC (Ser326) Antibody?

For maintaining antibody integrity and performance:

• Storage Temperature: Store at -20°C for up to 1 year from receipt

• Avoid Freeze-Thaw Cycles: Aliquot upon first thaw to minimize repeated freeze-thaw cycles

• Short-term Storage: For frequent use within one month, store at 4°C

• Formulation: The antibody is typically provided in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Working Dilutions: Prepare working dilutions fresh and use within the same day for optimal results

How can I use Phospho-NUDC (Ser326) Antibody to study cell cycle regulation and mitotic progression?

This antibody can be applied in several advanced experimental approaches:

• Time-course Analysis: Synchronize cells at different cell cycle stages and monitor NUDC Ser326 phosphorylation levels:

  • Use double thymidine block for G1/S boundary

  • RO-3306 for G2/M boundary

  • Nocodazole for prometaphase

  • Collect samples at defined intervals after release and analyze by Western blot

• Co-immunoprecipitation Studies: Use the antibody to:

  • Pull down Ser326-phosphorylated NUDC to identify interacting partners specific to this phosphorylated form

  • Investigate how phosphorylation affects protein complex formation during mitosis

• Chromatin Immunoprecipitation (ChIP): For analyzing recruitment of phosphorylated proteins to chromatin

• Immunofluorescence for Dynamics Analysis:

  • Perform double immunostaining with markers of mitotic structures (e.g., α-tubulin, γ-tubulin)

  • Analyze colocalization patterns through different mitotic phases

• Flow Cytometry: Combine with DNA content analysis to correlate phosphorylation levels with specific cell cycle stages

How can I investigate the relationship between PLK-mediated phosphorylation of NUDC and mitotic spindle formation?

To explore this relationship, consider these experimental approaches:

• PLK Inhibitor Studies:

  • Treat cells with PLK inhibitors (e.g., BI 2536, volasertib)

  • Analyze changes in NUDC Ser326 phosphorylation by Western blot

  • Simultaneously assess mitotic spindle morphology by immunofluorescence

• Phosphomimetic/Phosphodeficient Mutants:

  • Generate S326A (phosphodeficient) and S326D/E (phosphomimetic) NUDC mutants

  • Perform rescue experiments in NUDC-depleted cells

  • Compare mitotic phenotypes using live cell imaging

• Proximity Ligation Assay (PLA):

  • Detect in situ interactions between PLK and NUDC during different mitotic stages

  • Correlate with Ser326 phosphorylation status

• Immunoprecipitation-Kinase Assays:

  • Immunoprecipitate PLK from mitotic cells

  • Perform in vitro kinase assays with recombinant NUDC

  • Confirm phosphorylation at Ser326 using the phospho-specific antibody

What should I do if I experience high background in Western blots with Phospho-NUDC (Ser326) Antibody?

To reduce background and improve signal specificity:

• Blocking Optimization:

  • Test different blocking agents (5% BSA is often superior to milk for phospho-epitopes)

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

• Antibody Dilution:

  • Test a range of dilutions beyond the recommended 1:500-1:2000

  • Prepare antibody in fresh blocking buffer

• Washing Steps:

  • Increase number and duration of TBST/PBST washes (e.g., 5 x 10 minutes)

  • Use fresh washing buffers

• Phosphatase Inhibitors:

  • Ensure complete phosphatase inhibitor cocktails are used during sample preparation

  • Include specific inhibitors like sodium orthovanadate, sodium fluoride, and β-glycerophosphate

• Membrane Optimization:

  • Try PVDF instead of nitrocellulose or vice versa

  • Pre-equilibrate membrane in methanol followed by transfer buffer

• Secondary Antibody:

  • Use highly cross-adsorbed secondary antibodies

  • Reduce secondary antibody concentration

How can I optimize immunohistochemistry protocols for Phospho-NUDC (Ser326) Antibody?

For improved IHC results:

• Antigen Retrieval Optimization:

  • Compare heat-induced epitope retrieval methods:

    • Citrate buffer (pH 6.0)

    • EDTA buffer (pH 8.0-9.0)

    • Tris-EDTA (pH 9.0)

  • Test different retrieval times (10-30 minutes)

• Fixation Considerations:

  • Phospho-epitopes are sensitive to fixation conditions

  • Test samples fixed for different durations

  • For fresh samples, consider short (4-8 hours) paraformaldehyde fixation

• Antibody Incubation:

  • Compare room temperature (1-2 hours) vs. 4°C overnight incubation

  • Test the recommended dilution range (1:100-1:300)

• Signal Amplification:

  • Consider using polymer-based detection systems

  • For low abundance targets, try tyramide signal amplification

• Blocking Endogenous Activity:

  • Block endogenous peroxidase (3% H₂O₂, 10 minutes)

  • For biotin-based detection, include avidin/biotin blocking step

• Peptide Competition:

  • Include a peptide-blocked control section on your slide

How can I distinguish between specific and non-specific signals when using Phospho-NUDC (Ser326) Antibody?

For accurate data interpretation:

• Molecular Weight Verification:

  • NUDC has a calculated molecular weight of approximately 38 kDa

  • Confirm your band appears at the expected size

• Phosphatase Treatment Control:

  • The signal should disappear or significantly decrease after lambda phosphatase treatment

  • Compare band intensity quantitatively pre- and post-treatment

• Biological Validation:

  • Signal should increase during M phase when Ser326 phosphorylation is known to occur

  • Signal should decrease upon treatment with relevant kinase inhibitors

• Peptide Competition:

  • Pre-incubate antibody with phosphopeptide immunogen

  • Signal should be blocked by phosphopeptide but not by non-phosphorylated peptide

• Knockout/Knockdown Validation:

  • Test antibody in NUDC-depleted cells (siRNA or CRISPR)

  • Specific signals should be absent or significantly reduced

How should I quantify and normalize Phospho-NUDC (Ser326) levels in Western blots?

For reliable quantification:

• Total Protein Normalization:

  • Strip and reprobe membranes with total NUDC antibody

  • Calculate phospho-NUDC/total NUDC ratio

• Loading Control Selection:

  • Use housekeeping proteins (β-actin, GAPDH) with caution as their levels may vary

  • Consider total protein staining methods (Ponceau S, SYPRO Ruby)

• Multiple Internal Controls:

  • Include biological replicates from independent experiments

  • Test technical replicates within each experiment

• Linear Range Determination:

  • Perform dilution series to ensure detection is within linear range

  • Avoid overexposed bands which cannot be accurately quantified

• Densitometry Software:

  • Use software that can detect saturation (ImageJ, Image Lab)

  • Apply consistent background subtraction methods

What contradictions might arise in phosphorylation data and how can they be resolved?

Researchers may encounter these common data contradictions:

How can I effectively use the Cell-Based ELISA Kit for Phospho-NUDC (Ser326) in high-throughput screening?

The Cell-Based ELISA approach offers several advantages for screening applications:

• Experimental Setup:

  • Seed cells at consistent density in 96-well plates

  • Allow 24-48 hours for attachment before treatment

  • Include multiple technical replicates (minimum triplicate)

• Controls for Screening:

  • Positive control: Nocodazole-treated cells (M-phase enriched)

  • Negative control: Serum-starved cells (G0/G1 enriched)

  • Vehicle controls for all test compounds

• Normalization Strategies:

  • Normalize phospho-signal to total protein content

  • Include parallel wells for cell viability assessment

• Data Analysis:

  • Calculate Z-factor to assess assay quality

  • Apply appropriate statistical methods for hit identification

  • Cluster compounds by mechanism of action

• Hit Validation:

  • Confirm primary hits with dose-response curves

  • Validate with orthogonal assays (Western blot, IF)

  • Assess effects on other mitotic phosphoproteins

The assay can detect changes in NUDC phosphorylation in response to treatments directly in cultured cells without the need for lysate preparation, making it ideal for screening compound libraries or siRNA panels .

What considerations are important when using Phospho-NUDC (Ser326) Antibody for immunofluorescence studies of mitotic cells?

For optimal immunofluorescence visualization:

• Cell Preparation:

  • Consider growing cells on coated coverslips for better attachment during mitosis

  • Test different fixation methods (4% PFA, methanol, or combinations)

  • Use extraction buffers containing 0.5% Triton X-100 before fixation to remove cytoplasmic proteins

• Co-staining Recommendations:

  • Combine with α-tubulin staining to visualize mitotic spindles

  • Include DNA staining (DAPI, Hoechst) to identify mitotic stages

  • Consider γ-tubulin for centrosome visualization

• Imaging Parameters:

  • Use confocal microscopy for better spatial resolution

  • Capture z-stacks to fully visualize 3D structures

  • Apply deconvolution for improved signal-to-noise ratio

• Controls:

  • Include peptide competition control

  • Stain synchronized populations (prometaphase, metaphase, anaphase)

  • Include phosphatase-treated samples as negative controls

• Quantification Approaches:

  • Measure fluorescence intensity across different mitotic phases

  • Assess colocalization with mitotic structures

  • Track phosphorylation dynamics in time-lapse experiments

The recommended dilution range for immunofluorescence is 1:50-200, but optimization may be required for specific experimental conditions .

How can Phospho-NUDC (Ser326) Antibody be used to study cell division abnormalities in cancer models?

This antibody can be valuable for cancer research in several contexts:

• Tumor Tissue Analysis:

  • Compare phosphorylation patterns between normal and tumor tissues

  • Correlate with markers of proliferation (Ki-67, PCNA)

  • Assess relationship with chromosomal instability markers

• Cancer Cell Line Panels:

  • Screen phosphorylation status across cell lines with different aggressiveness

  • Correlate with mitotic index and chromosome segregation errors

  • Link to sensitivity to anti-mitotic drugs

• Drug Response Studies:

  • Monitor changes in phosphorylation after treatment with:

    • Microtubule-targeting agents (taxanes, vinca alkaloids)

    • Mitotic kinase inhibitors (PLK, Aurora kinases)

    • Cell cycle checkpoint inhibitors

• Genetic Manipulation Models:

  • Assess effects of oncogene activation or tumor suppressor loss

  • Create phosphomimetic/phosphodeficient NUDC mutants in cancer cells

  • Evaluate impact on chromosomal stability and cell proliferation

• Patient-Derived Xenografts (PDX):

  • Monitor phosphorylation in PDX models before and after treatment

  • Correlate with treatment response and resistance development

This research could help establish whether aberrant NUDC phosphorylation contributes to mitotic defects in cancer and whether it could serve as a potential biomarker or therapeutic target.

What techniques can be used to simultaneously assess multiple phosphorylation sites on NUDC protein?

For comprehensive phosphorylation analysis:

• Multiplexed Western Blotting:

  • Sequential probing with different phospho-specific antibodies

  • Use fluorescently-labeled secondary antibodies with different spectra

  • Include total NUDC antibody for normalization

• Mass Spectrometry Approaches:

  • Immunoprecipitate NUDC and analyze by phospho-proteomics

  • Use targeted methods like Multiple Reaction Monitoring (MRM)

  • Apply phospho-enrichment techniques (TiO₂, IMAC)

• Phospho-Peptide Arrays:

  • Design peptide arrays covering multiple NUDC phosphorylation sites

  • Test kinase activity on these peptides

  • Validate findings with site-specific antibodies

• Proximity Ligation Assay (PLA):

  • Combine antibodies against different phospho-sites

  • Visualize coincident phosphorylation events in situ

• ELISA-Based Multiplexing:

  • Use multiple wells with different capture antibodies

  • Develop sandwich ELISA approaches for different phospho-sites

Remember that NUDC is phosphorylated at multiple sites, including Ser-274 and Ser-326, both of which are necessary for correct mitotic spindle formation and chromosome separation .

What are emerging applications for Phospho-NUDC (Ser326) Antibody in studying neurodegenerative diseases?

Given NUDC's role in neurogenesis and neuronal migration , this antibody holds potential for neurodegenerative disease research:

• Neuronal Differentiation Studies:

  • Monitor NUDC phosphorylation during neuronal differentiation from stem cells

  • Assess impact on neuronal migration and positioning

  • Correlate with cytoskeletal dynamics in neurite outgrowth

• Neurodegeneration Models:

  • Investigate phosphorylation changes in models of:

    • Alzheimer's disease

    • Parkinson's disease

    • Amyotrophic Lateral Sclerosis (ALS)

  • Correlate with mitochondrial transport defects

• Brain Development:

  • Analyze phosphorylation patterns during cortical development

  • Study impact on interkinetic nuclear migration

  • Assess relationship with neuronal positioning defects

• Neuronal Stress Responses:

  • Monitor phosphorylation changes after oxidative stress

  • Study relationship with neuronal cell cycle re-entry (pathological in mature neurons)

  • Correlation with neuronal cell death pathways

• Therapeutic Screening:

  • Use to evaluate neuroprotective compounds that may influence NUDC function

  • Screen for compounds that normalize aberrant phosphorylation

These applications could help establish whether dysregulation of NUDC phosphorylation contributes to neuronal vulnerability in disease states.

How might technological advances improve the utility of phospho-specific antibodies like Phospho-NUDC (Ser326) Antibody?

Several emerging technologies could enhance research with phospho-specific antibodies:

• Single-Cell Western Blotting:

  • Analyze phosphorylation heterogeneity in individual cells

  • Correlate with cell cycle position at single-cell resolution

  • Combine with other single-cell analyses (RNA-seq, ATAC-seq)

• Super-Resolution Microscopy:

  • Visualize phospho-protein localization at nanometer resolution

  • Study co-localization with mitotic structures more precisely

  • Track dynamic changes during mitotic progression

• Microfluidic Platforms:

  • Develop lab-on-a-chip approaches for phosphorylation analysis

  • Enable real-time monitoring of phosphorylation in living cells

  • Combine with other assays for multiparameter analysis

• CRISPR-Based Approaches:

  • Generate knock-in cell lines with fluorescent tags near phosphorylation sites

  • Create cellular biosensors for NUDC phosphorylation

  • Apply base editing to generate phosphomimetic mutations

• Affinity Enhancement:

  • Engineer antibodies with improved affinity and specificity

  • Develop novel scaffolds beyond traditional antibodies

  • Create bispecific reagents recognizing multiple phospho-sites