Phospho-NIFK (T234) Antibody

What is NIFK and what cellular functions does it regulate?

NIFK (nucleolar protein interacting with the FHA domain of MKI67) encodes a protein that specifically interacts with the forkhead-associated domain of the Ki-67 antigen. Research indicates that NIFK likely plays critical roles in several cellular processes, particularly cell division and proliferation. The protein has RNA-binding capabilities and appears to be significantly involved in mitosis and cell cycle progression . Genomic studies have identified multiple NIFK pseudogenes distributed across chromosomes 5, 10, 12, 15, and 19, suggesting evolutionary importance of this gene family . The phosphorylation status of NIFK, particularly at the Thr234 position, appears to regulate its activity during mitotic processes, making phospho-specific antibodies valuable tools for studying its function in normal and disease states.

What is the molecular weight of NIFK and how does phosphorylation affect it?

When analyzing NIFK using western blotting techniques, researchers should note the discrepancy between the calculated and observed molecular weights. While the calculated molecular weight of NIFK is approximately 34.2 kDa, it typically appears at approximately 72 kDa on SDS-PAGE gels . This significant difference (approximately 37.8 kDa) can be attributed to post-translational modifications, particularly phosphorylation, and the protein's structural characteristics. Phosphorylation at Thr234 represents one of several potential phosphorylation sites that collectively contribute to the observed mobility shift. When designing experiments, researchers should account for this discrepancy to accurately identify NIFK bands in western blot analyses.

What experimental applications are validated for Phospho-NIFK (T234) antibodies?

Phospho-NIFK (T234) antibodies have been validated for multiple experimental applications with specific recommended dilutions based on rigorous validation studies. The primary applications include:

These applications have been experimentally verified to produce specific signal with minimal background when proper controls are employed . The antibody has been confirmed to react specifically with human and mouse NIFK proteins phosphorylated at Thr234, making it suitable for comparative studies across these species .

How can I distinguish between phosphorylated and non-phosphorylated forms of NIFK in experimental systems?

Distinguishing between phosphorylated and non-phosphorylated forms of NIFK requires careful experimental design and appropriate controls. The Phospho-NIFK (T234) antibody specifically recognizes NIFK only when phosphorylated at threonine 234, enabling selective detection of this phosphorylated form .

To confirm specificity and distinguish between phosphorylated and non-phosphorylated forms, implement the following methodological approach:

• Use paired phospho-specific and total NIFK antibodies in parallel experiments

• Include phosphatase treatment controls where samples are incubated with lambda phosphatase prior to analysis

• Utilize competing peptide controls with both phosphorylated and non-phosphorylated peptides

For western blot applications, the phosphorylated form may display subtle mobility shifts compared to the non-phosphorylated form. In dot blot assays, as demonstrated with similar phospho-specific antibodies, the antibody should show strong reactivity with the phosphorylated peptide but minimal to no reactivity with the non-phosphorylated peptide . This differential reactivity pattern confirms the phospho-specificity of the antibody and validates experimental findings.

What is the relationship between NIFK phosphorylation and the Ki-67 protein during cell cycle progression?

NIFK interacts specifically with the forkhead-associated (FHA) domain of Ki-67, a well-established proliferation marker used in cancer diagnostics . The phosphorylation of NIFK at Thr234 likely modulates this interaction during different phases of the cell cycle. Current research suggests a model wherein:

• During interphase, NIFK may remain largely unphosphorylated at Thr234

• As cells enter mitosis, phosphorylation at Thr234 increases, potentially altering the interaction with Ki-67

• This phosphorylation-dependent interaction may regulate nucleolar disassembly and reassembly during mitosis

To effectively study this relationship, researchers should design time-course experiments throughout the cell cycle using synchronized cell populations. Co-immunoprecipitation experiments using the Phospho-NIFK (T234) antibody can help identify phosphorylation-dependent protein complexes. Confocal microscopy with dual staining for phospho-NIFK and Ki-67 can reveal spatial and temporal changes in their localization and interaction during mitotic progression.

How does NIFK phosphorylation compare with other cell cycle-regulated phosphorylation events?

The phosphorylation of NIFK at Thr234 represents one of many coordinated phosphorylation events that regulate the cell cycle. Comparative analysis with other phosphorylation events provides context for understanding NIFK's role in the broader cell cycle regulation network.

Similar to MARK proteins (which undergo phosphorylation at sites like T214 in MARK4, T208 in MARK2, and T234 in MARK3) and CaMKII proteins (phosphorylated at T286) , NIFK phosphorylation likely occurs within specific sequence motifs recognized by cell cycle-regulated kinases. The timing and functional consequences of these events collectively orchestrate mitotic progression.

For comprehensive cell cycle studies, researchers should consider employing multiplexed approaches that simultaneously examine several phosphorylation events, including:

• Phospho-NIFK (T234)

• Cyclin-dependent kinase substrates

• Aurora kinase substrates

• Polo-like kinase substrates

This integrated approach can reveal the temporal sequence of phosphorylation events and their functional interdependencies during cell cycle progression.

What are the optimal sample preparation protocols for detecting phospho-NIFK in different experimental systems?

Successful detection of phosphorylated NIFK requires careful attention to sample preparation to preserve phosphorylation status. The following protocols are recommended based on application type:

For Western Blotting:

• Rapidly harvest cells or tissues and immediately lyse in ice-cold buffer containing phosphatase inhibitors (10 mM sodium fluoride, 2 mM sodium orthovanadate, 2 mM sodium pyrophosphate, and 1× phosphatase inhibitor cocktail)

• Maintain samples at 4°C throughout processing

• Add SDS sample buffer and heat at 95°C for 5 minutes

• Load 20-50 μg of total protein per lane

• Use freshly prepared transfer buffers without methanol for optimal transfer of phosphoproteins

For Immunohistochemistry:

• Fix tissues in 4% paraformaldehyde for no more than 24 hours

• Use citrate buffer (pH 6.0) for antigen retrieval

• Block with 5% BSA in TBS with 0.1% Tween-20 for 1 hour

• Incubate with Phospho-NIFK (T234) antibody at 1:100-1:300 dilution overnight at 4°C

• Include phosphatase-treated serial sections as negative controls

For Immunofluorescence:

• Fix cells in 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 for 5 minutes

• Block with 3% BSA for 30 minutes

• Incubate with antibody at 1:200-1:1000 dilution for 1-2 hours

• Co-stain with markers for cell cycle phases to correlate phosphorylation with cell cycle position

These protocols have been optimized to maintain phosphorylation status while providing clear signal with minimal background.

How can I validate the specificity of phospho-NIFK (T234) antibodies in my experimental system?

Validating antibody specificity is crucial for obtaining reliable results. For phospho-NIFK (T234) antibodies, implement the following validation approaches:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess phosphorylated and non-phosphorylated peptides

  • Only the phosphorylated peptide should block antibody binding

  • Compare staining patterns with and without competing peptides

• Phosphatase Treatment Controls:

  • Treat duplicate samples with lambda phosphatase to remove phosphate groups

  • The phospho-specific antibody should show diminished or absent signal in treated samples

• Genetic Validation:

  • Use CRISPR/Cas9 to generate NIFK knockout cells as negative controls

  • Create T234A mutant (non-phosphorylatable) and T234D mutant (phosphomimetic) constructs

  • Compare antibody reactivity across these genetic models

• Site-Specific Mutagenesis:

  • Express wild-type NIFK and T234A mutant constructs

  • The antibody should detect only the wild-type protein when phosphorylated

  • This approach confirms the exact epitope recognized by the antibody

Similar validation approaches have been demonstrated with other phospho-specific antibodies, such as those against phosphorylated MARK proteins, where dot blot assays clearly differentiated between phosphorylated and non-phosphorylated peptides .

What controls should be included when using phospho-NIFK (T234) antibody in cell cycle research?

Rigorous experimental design requires appropriate controls to ensure reliable interpretation of results involving phospho-NIFK (T234) antibody:

For cell synchronization experiments, include time-course controls to track phosphorylation changes as cells progress through the cell cycle. When using chemical synchronization methods (e.g., nocodazole, thymidine block), include vehicle-treated controls to account for potential artifacts induced by the synchronization agents themselves.

Why might I observe inconsistent results when detecting phospho-NIFK across different cell lines?

Inconsistent detection of phospho-NIFK (T234) across cell lines can stem from multiple biological and technical factors:

• Cell Line-Specific Expression Levels:

  • NIFK expression varies significantly across cell types

  • Proliferation rates affect the proportion of cells with phosphorylated NIFK

  • Verify total NIFK levels before interpreting phosphorylation differences

• Differential Phosphorylation Regulation:

  • Cell type-specific kinase and phosphatase activities affect phosphorylation levels

  • Growth conditions and cell density influence phosphorylation status

  • Standardize culture conditions and harvest timing

• Technical Considerations:

  • Cell lysis methods may differentially preserve phosphorylation

  • Sample buffer composition affects epitope accessibility

  • Antibody concentration may need optimization for each cell line

To address these issues, perform careful titration experiments with the antibody using a dilution series from 1:50 to 1:1000 for each cell line. Additionally, standardize cell culture conditions, ensuring consistent cell cycle distribution across samples through flow cytometry analysis of parallel cultures.

How can phospho-NIFK (T234) antibody be incorporated into multiplexed imaging systems?

Integrating phospho-NIFK (T234) antibody into multiplexed imaging requires careful planning to avoid antibody cross-reactivity and signal interference:

• Sequential Immunostaining Approach:

  • Use tyramide signal amplification (TSA) with spectral unmixing

  • Apply antibodies sequentially with microwave-based antibody stripping between rounds

  • Begin with phospho-NIFK antibody application, as phospho-epitopes are more sensitive to degradation

• Antibody Conjugation Strategies:

  • Directly label phospho-NIFK antibody with bright, photostable fluorophores

  • Select fluorophores with minimal spectral overlap

  • Use quantum dots for enhanced stability in sequential imaging

• Complementary Markers:

  • Co-stain with cell cycle markers (Ki-67, PCNA)

  • Include nuclear/nucleolar markers (fibrillarin, nucleolin)

  • Add cytoskeletal markers to assess mitotic structures

Similar multiplexing approaches have been successfully employed in other antibody screening systems, such as the in vivo screening method for recombinant monoclonal antibodies described in the eLife study , which could be adapted for phospho-specific antibody applications.

Can computational approaches help interpret phospho-NIFK (T234) data in the context of cell cycle regulation?

Computational methods significantly enhance the analysis and interpretation of phospho-NIFK data within the broader context of cell cycle regulation:

• Quantitative Image Analysis:

  • Develop automated segmentation algorithms to identify subcellular compartments

  • Implement pixel-based colocalization analysis with other cell cycle markers

  • Apply machine learning approaches to classify cells by cycle phase based on phospho-NIFK patterns

• Temporal Dynamics Modeling:

  • Create mathematical models incorporating phosphorylation/dephosphorylation kinetics

  • Simulate the effects of perturbing kinase or phosphatase activities

  • Predict phospho-NIFK behavior under various experimental conditions

• Network Analysis:

  • Integrate phospho-NIFK data with phosphoproteomic datasets

  • Construct signaling networks connecting NIFK to other cell cycle regulators

  • Identify feedback loops and regulatory mechanisms

• Single-Cell Analysis Workflows:

  • Correlate phospho-NIFK levels with cell morphology and cell cycle position

  • Account for cell-to-cell heterogeneity in asynchronous populations

  • Develop trajectory inference methods to map phosphorylation dynamics

These computational approaches transform static phospho-NIFK measurements into dynamic insights about cell cycle progression and regulation, similar to approaches that have been applied to other phospho-specific antibody systems .

What are emerging applications for phospho-NIFK (T234) antibodies in cancer research?

Phospho-NIFK (T234) antibodies hold significant potential for advancing cancer research through several innovative applications:

• Biomarker Development:

  • Assess phospho-NIFK levels in patient-derived samples

  • Correlate with clinical outcomes and treatment responses

  • Develop immunohistochemistry-based prognostic assays

• Therapeutic Target Validation:

  • Screen for kinase inhibitors that modulate NIFK phosphorylation

  • Evaluate effects on cell proliferation and survival

  • Assess combination therapies targeting multiple phosphorylation events

• Cell Cycle Checkpoint Studies:

  • Examine phospho-NIFK dynamics following DNA damage

  • Investigate relationships with checkpoint proteins (ATM/ATR, CHK1/2)

  • Identify potential synthetic lethal interactions with checkpoint inhibitors

These applications build upon the foundational understanding that NIFK interacts with Ki-67, a well-established proliferation marker, and that its phosphorylation status likely influences cell cycle progression . Given NIFK's potential role in mitosis and proliferation, characterizing its phosphorylation in cancer contexts could reveal new therapeutic vulnerabilities.

How might new antibody development technologies improve phospho-specific detection systems?

The field of phospho-specific antibody development continues to advance, with several promising technologies that could enhance phospho-NIFK detection:

• Genotype-Phenotype Linked Antibody Systems:

  • Novel screening methods, such as the Golden Gate-based dual-expression vector system described in recent research , enable rapid identification of high-affinity, phospho-specific antibodies

  • These systems facilitate the generation of monoclonal antibodies with enhanced specificity compared to polyclonal antibodies

• Synthetic Antibody Technologies:

  • Phage display libraries specifically designed for phospho-epitope recognition

  • Structure-guided antibody engineering to enhance specificity

  • Computational design of complementarity-determining regions (CDRs) optimized for phospho-epitope binding

• Nanobody and Alternative Scaffold Technologies:

  • Single-domain antibody fragments offer improved tissue penetration

  • Smaller binding footprints may provide enhanced discrimination between phosphorylated and non-phosphorylated epitopes

  • Engineered protein scaffolds with customized binding surfaces for phospho-epitope recognition

These technological advances promise to develop next-generation phospho-specific antibodies with improved sensitivity, specificity, and consistency across diverse experimental applications.

What standardization efforts are needed for phospho-specific antibodies in the research community?

Standardization represents a critical need in the phospho-specific antibody field to ensure reproducibility and reliability of research findings:

• Validation Standards:

  • Establish minimum validation criteria specifically for phospho-antibodies

  • Implement standardized reporting of validation experiments

  • Create shared repositories of validation data accessible to researchers

• Reference Materials:

  • Develop certified reference standards for phospho-NIFK

  • Create standard operating procedures for sample preparation

  • Establish interlaboratory testing programs

• Data Sharing Initiatives:

  • Create public databases of antibody performance across different applications

  • Implement standardized formats for sharing raw data

  • Develop open-source analysis pipelines for phospho-specific antibody experiments