NIFK Antibody

What is NIFK and why is it significant in research?

NIFK is a nucleolar phosphoprotein that interacts with the FHA domain of Ki-67, a well-established proliferation marker. Research has demonstrated that NIFK enhances Ki-67-dependent proliferation and promotes migration, invasion in vitro, and metastasis in vivo via downregulation of casein kinase 1α (CK1α) . NIFK functions through the Runx-1-dependent repression of CK1α expression, which subsequently activates TCF/β-catenin signaling . High NIFK expression correlates with poor prognosis in several cancer types, including lung, breast, and colorectal cancers, making it an important target for understanding cancer mechanisms and potential therapeutic interventions .

What types of NIFK antibodies are available and how should I choose between them?

Several types of NIFK antibodies are available for research:

When selecting an antibody:

• Choose monoclonal antibodies for highly specific detection and reproducible results across experiments

• Select polyclonal antibodies for enhanced sensitivity, particularly in applications where protein conformation may be altered

• Consider validated applications (WB, IHC, IF/ICC) based on your experimental needs

• Review validation data demonstrating reactivity with your species of interest (most NIFK antibodies are validated for human samples)

What are the optimal protocols for using NIFK antibodies in Western Blotting?

For optimal Western Blot detection of NIFK:

• Sample preparation:

  • Use validated cell lines that express NIFK (HeLa, HEK-293, HepG2)

  • Prepare protein lysates with protease inhibitors

• Electrophoresis and transfer:

  • Load 20-50 μg protein per lane

  • Use 10-12% SDS-PAGE gel

  • Transfer to PVDF or nitrocellulose membrane

• Antibody incubation:

  • Block with 5% non-fat milk in TBST

  • Dilute primary antibody appropriately (1:1000-1:8000 for polyclonal)

  • Incubate overnight at 4°C

  • Wash thoroughly with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody

• Detection and analysis:

  • Develop using ECL substrate

  • Expected molecular weight: 34 kDa

  • Include positive controls (K562 cells) and negative controls (NIFK knockdown cells)

What are the recommended protocols for immunohistochemistry detection of NIFK?

For IHC detection of NIFK in tissue samples:

• Tissue preparation:

  • Fix in formalin and embed in paraffin

  • Section at 4-5 μm thickness

  • Mount on positively charged slides

• Pretreatment:

  • Deparaffinize and rehydrate

  • Perform antigen retrieval using TE buffer pH 9.0 or citrate buffer pH 6.0

  • Block endogenous peroxidase activity with 3% hydrogen peroxide

  • Block non-specific binding with serum

• Antibody incubation:

  • Dilute primary antibody appropriately (1:500-1:2000 for IHC)

  • Incubate overnight at 4°C

  • Apply appropriate secondary antibody and detection system

• Validated positive controls:

  • Human liver cancer tissue

  • Human renal cell carcinoma tissue

How do I optimize immunofluorescence staining for NIFK?

For optimal immunofluorescence detection of NIFK:

• Cell preparation:

  • Culture cells on coverslips to 70-80% confluence

  • Fix with 4% paraformaldehyde (15 minutes, room temperature)

  • Permeabilize with 0.1-0.5% Triton X-100 in PBS (10 minutes)

• Antibody incubation:

  • Block with 5% normal serum

  • Dilute primary antibody appropriately (1:50-1:500 for IF/ICC)

  • Incubate overnight at 4°C or 2 hours at room temperature

  • Wash thoroughly with PBS

  • Incubate with fluorophore-conjugated secondary antibody

• Visualization:

  • Counterstain nuclei with DAPI

  • Mount using anti-fade mounting medium

  • NIFK typically shows nucleolar localization pattern

• Validated cell lines:

  • MCF-7, U251, U2OS cells have been successfully used

How can I effectively use NIFK antibodies to study its role in cancer progression?

To investigate NIFK's role in cancer progression:

What are the common troubleshooting strategies for weak or absent NIFK signals?

When troubleshooting weak or absent NIFK antibody signals:

• Validate antibody functionality:

  • Test with positive control cell lines (HeLa, HEK-293, HepG2)

  • Verify antibody storage conditions (most require -20°C storage)

  • Check antibody expiration date

• Optimize sample preparation:

  • For fixed tissues, enhance antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • For cells, adjust fixation and permeabilization conditions

  • Ensure protein isn't degraded in lysates (use fresh protease inhibitors)

• Adjust antibody conditions:

  • Test concentration gradient (WB: 1:1000-1:8000; IHC: 1:500-1:2000; IF: 1:50-1:500)

  • Extend primary antibody incubation time

  • Try alternative blocking reagents (BSA vs. serum)

• Enhance signal detection:

  • Implement signal amplification methods (biotin-streptavidin, tyramide)

  • Increase exposure time for WB

  • Optimize microscope settings for IF/IHC

• Consider alternative antibodies:

  • Try antibodies targeting different NIFK epitopes

  • Switch between monoclonal and polyclonal options

How can I quantify NIFK expression in tissue samples for clinical correlations?

For accurate quantification of NIFK expression in tissues:

• Standardize IHC protocols:

  • Use consistent antibody dilutions (1:500-1:2000)

  • Standardize incubation times and detection systems

  • Process all samples in a single batch when possible

• Include appropriate controls:

  • Positive and negative tissue controls in each staining batch

  • Internal control tissues within each slide when possible

• Implement digital image analysis:

  • Capture images under standardized conditions

  • Use software like ImageJ or QuPath for quantification

  • Establish regions of interest (tumor areas vs. stroma)

• Select appropriate scoring methods:

  • H-score (combining intensity and percentage positive cells)

  • Allred score

  • Digital quantification of staining intensity

• Validate methodology:

  • Compare results using different NIFK antibodies

  • Correlate with other detection methods (e.g., WB or qPCR)

• Statistical analysis:

  • Compare NIFK expression across different patient groups

  • Correlate with clinical parameters (stage, grade, survival)

  • Perform multivariate analysis to assess independent prognostic value

What is known about the relationship between NIFK and Ki-67 in cellular proliferation?

The relationship between NIFK and Ki-67 involves several key mechanisms:

• Physical interaction:

  • NIFK (Nucleolar protein interacting with the FHA domain of pKI-67) directly interacts with the FHA domain of Ki-67

  • This interaction occurs primarily in the nucleolus

• Functional relationship:

  • Ki-67 serves as a marker for cellular proliferation, expressed during all active cell cycle phases (G1, S, G2, mitosis) but absent in quiescent cells

  • NIFK enhances Ki-67-dependent proliferation, suggesting a role in regulating proliferative capacity

• Cancer implications:

  • Both Ki-67 and NIFK are overexpressed in multiple cancer types

  • Ki-67 expression levels serve as a proliferation index in clinical settings

  • NIFK expression correlates with poor prognosis, potentially through enhancement of Ki-67-mediated proliferation

• Mechanistic insights:

  • Studies suggest that NIFK influences nucleolar function and ribosome biogenesis

  • The NIFK-Ki-67 interaction may regulate cell cycle progression through nucleolar signaling pathways

Research methods for studying this relationship include co-immunoprecipitation, co-localization studies using immunofluorescence, and functional assays examining proliferation after modulating NIFK expression.

How does NIFK expression correlate with metastatic potential in cancer models?

NIFK expression demonstrates significant correlations with metastatic potential:

These findings establish NIFK as both a prognostic biomarker and potential therapeutic target in cancer progression.

What is the significance of NIFK subcellular localization in different cell types or disease states?

The subcellular localization of NIFK provides important insights:

• Normal localization pattern:

  • NIFK primarily localizes to the nucleolus in normal cells

  • Functions as a nucleolar marker in immunofluorescence studies

  • Co-localizes with other nucleolar proteins involved in ribosome biogenesis

• Visualization methods:

  • Immunofluorescence with NIFK antibodies shows specific staining of nucleoli

  • Can be visualized alongside cytoskeletal elements (e.g., microtubules) to demonstrate nuclear/nucleolar specificity

  • Validated in multiple cell lines including MCF-7, U251, and U2OS cells

• Changes in disease states:

  • Alterations in nucleolar morphology and function are hallmarks of cancer

  • NIFK localization patterns may reflect changes in cellular proliferation status

  • Potential redistribution during cell cycle progression or cellular stress

• Functional implications:

  • Nucleolar localization consistent with roles in ribosome biogenesis and proliferation

  • Interaction with Ki-67 occurs primarily in the nucleolus

  • Changes in localization may correlate with activation of specific signaling pathways

Understanding NIFK's subcellular distribution provides insights into its biological functions and potential roles in disease processes.

How can NIFK antibodies be used in combination with other markers for comprehensive cancer profiling?

For comprehensive cancer profiling using NIFK antibodies:

• Multiplexed immunofluorescence panels:

  • Combine NIFK with Ki-67 (proliferation marker)

  • Include markers for β-catenin pathway components (based on CK1α/β-catenin pathway connection)

  • Add cell-type specific markers to identify NIFK expression in tumor vs. stromal compartments

• Sequential staining approaches:

  • Perform NIFK staining on serial tissue sections alongside other markers

  • Use digital alignment tools to correlate expression patterns across sections

  • Quantify co-expression at the single-cell level

• Flow cytometry applications:

  • Combine NIFK antibodies with cell cycle markers

  • Include apoptosis markers to correlate NIFK expression with cell survival

  • Sort NIFK-high vs. NIFK-low populations for functional studies

• Technical considerations:

  • Ensure antibody compatibility (different host species or directly conjugated)

  • Optimize staining protocols for each marker individually before combining

  • Include appropriate controls to assess specificity of each signal

• Analysis approaches:

  • Implement machine learning algorithms for pattern recognition

  • Quantify co-localization coefficients for spatial relationships

  • Correlate expression patterns with clinical outcomes

What novel applications are emerging for NIFK antibodies in biomedical research?

Emerging applications for NIFK antibodies include:

• Liquid biopsy development:

  • Detection of circulating tumor cells expressing NIFK

  • Correlation with metastatic potential and disease progression

  • Potential for monitoring treatment response

• Therapeutic target validation:

  • Screening for compounds that disrupt NIFK-Ki-67 interaction

  • Evaluation of drugs targeting CK1α/β-catenin pathway in NIFK-high tumors

  • Development of antibody-drug conjugates for targeted therapy

• Advanced imaging approaches:

  • Super-resolution microscopy to examine NIFK's precise nucleolar localization

  • Live-cell imaging with fluorescently tagged antibody fragments

  • Correlative light and electron microscopy for ultrastructural studies

• Single-cell applications:

  • Integration with single-cell transcriptomics

  • Mass cytometry (CyTOF) for high-dimensional protein analysis

  • Spatial transcriptomics to correlate NIFK protein expression with local gene expression

• Antibody engineering applications:

  • Development of recombinant antibody fragments (scFvs, Fabs) for specialized applications

  • Creation of bispecific antibodies targeting NIFK and related cancer markers

  • Similar to approaches used in antibody development described for SARS-CoV-2

These emerging applications reflect the growing importance of NIFK as a biomarker and potential therapeutic target in cancer research.