narfl Antibody

What is NARFL and why is it important in biomedical research?

NARFL (Nuclear prelamin A recognition factor-like protein), also known as CIAO3, PRN, or IOP1, plays a critical role in the cytosolic iron-sulfur cluster assembly pathway. This protein is essential for the maturation of extramitochondrial sulfur and iron proteins, which are vital for numerous enzymatic reactions . Research has demonstrated that NARFL deficiency impairs mitochondrial integrity and function, potentially affecting various cellular processes including energy metabolism and oxidative stress regulation . The NARFL gene produces three alternatively spliced isoforms, highlighting its versatility in cellular systems . Recent studies have established connections between NARFL and hypoxia-inducible factor 1-alpha (HIF-1α) signaling, suggesting its involvement in oxygen sensing pathways that are critical in cancer biology and development .

What types of NARFL antibodies are available for research applications?

Researchers have access to several types of NARFL antibodies optimized for different experimental applications:

• Polyclonal antibodies: Products like 13652-1-AP target NARFL in Western blot, immunofluorescence, immunohistochemistry, and ELISA applications, showing reactivity with human, mouse, and rat samples . These are typically produced in rabbits immunized with NARFL fusion proteins or fragments .

• Monoclonal antibodies: The E-7 mouse monoclonal IgG2a kappa light chain antibody detects NARFL protein of mouse, rat, and human origin across multiple applications including Western blotting, immunoprecipitation, immunofluorescence, and ELISA .

• Conjugated antibodies: Various conjugated forms are available, including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and Alexa Fluor conjugates, facilitating different detection methods .

• Specialized detection antibodies: Some antibodies like NARFL Antibody [CoraFluor™ 1] incorporate advanced fluorescent technologies optimized for applications such as Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assays .

How should researchers choose between monoclonal and polyclonal NARFL antibodies?

The choice between monoclonal and polyclonal NARFL antibodies should be based on experimental objectives:

Monoclonal antibodies (e.g., NARFL Antibody E-7) offer:

• Higher specificity for a single epitope

• Lower batch-to-batch variability

• Superior performance in applications requiring high reproducibility

• Better suited for quantitative analyses and specific protein domain targeting

Polyclonal antibodies (e.g., 13652-1-AP) provide:

• Recognition of multiple epitopes on the NARFL protein

• Stronger signal due to binding multiple sites on each target molecule

• Greater tolerance to protein denaturation or conformational changes

• Better performance in applications where native protein detection is challenging

For detecting small amounts of NARFL in samples or when protein may be partially denatured, polyclonal antibodies often deliver better results. For highly specific detection of particular NARFL isoforms or when absolute epitope specificity is required, monoclonal antibodies are preferable . If immunoprecipitation is a primary application, the NARFL Antibody (E-7) has documented effectiveness for this purpose .

What are the optimal protocols for NARFL detection by Western blotting?

Based on validated protocols for NARFL antibodies, researchers should consider the following approach:

• Sample preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • Use ~20-40 μg of total protein per lane

  • Observe that NARFL typically appears at 26-28 kDa (observed) versus the calculated molecular weight of 53 kDa (476 aa)

• Antibody dilutions:

  • For polyclonal antibodies like 13652-1-AP: 1:1000-1:4000 dilution

  • For monoclonal antibodies like E-7: Optimal dilution should be experimentally determined for each application

• Detection systems:

  • Both chemiluminescence and fluorescence-based systems are compatible

  • For multiplexing experiments, consider using antibodies with different conjugates

• Controls:

  • Include positive controls (HeLa and HEK-293 cells express detectable levels of NARFL)

  • Include NARFL knockdown samples as negative controls where possible

Note that NARFL has three isoforms produced by alternative splicing, which may complicate band pattern interpretation . Research has shown that knockdown of NARFL in cell lines such as A549 and H1299 significantly reduces protein levels, making these suitable models for validation studies .

What are the best practices for immunohistochemical detection of NARFL?

For optimal immunohistochemical detection of NARFL in tissue sections:

• Tissue preparation:

  • Both formalin-fixed paraffin-embedded (FFPE) and frozen sections can be used

  • For FFPE sections, antigen retrieval is critical - suggested methods include:

    • TE buffer pH 9.0 (preferred)

    • Citrate buffer pH 6.0 (alternative)

• Antibody concentrations:

  • For polyclonal antibodies like 13652-1-AP: 1:50-1:500 dilution range

  • For other antibodies: Perform dilution series to determine optimal concentration

• Detection systems:

  • Both chromogenic and fluorescent detection systems are compatible

  • For tissue with high autofluorescence, chromogenic detection may be preferable

• Positive control tissues:

  • Human colon tissue has been validated as a positive control for NARFL staining

  • Include known positive controls in each staining batch

• Counterstaining:

  • Use appropriate nuclear counterstains to facilitate localization of NARFL signals

  • Be aware that NARFL may show both cytoplasmic and nuclear localization

When studying NARFL in lung cancer samples, researchers should note that NARFL deficiency has been associated with poor survival rates in NSCLC patients, making proper quantification of staining intensity particularly important .

How can researchers troubleshoot non-specific binding issues with NARFL antibodies?

When encountering non-specific binding with NARFL antibodies, consider these methodological solutions:

• For Western blot applications:

  • Increase blocking time (5% non-fat milk or BSA for 1-2 hours)

  • Optimize primary antibody dilution (test ranges between 1:1000-1:4000)

  • Increase washing duration and frequency (5-6 washes of 5-10 minutes each)

  • Consider alternative blocking reagents if background persists

  • Remember that NARFL observed molecular weight (26-28 kDa) differs significantly from calculated (53 kDa)

• For immunohistochemistry:

  • Increase blocking time and concentration

  • Optimize antibody dilution (starting with 1:50-1:500 range)

  • Include additional washing steps before and after primary antibody incubation

  • Consider using specialized blocking solutions containing IgG or serum matched to secondary antibody species

  • Test alternative antigen retrieval methods (compare TE buffer pH 9.0 versus citrate buffer pH 6.0)

• For all applications:

  • Validate specificity using NARFL knockdown or knockout controls

  • Consider pre-absorbing the antibody with the immunogen if available

  • Test multiple NARFL antibodies targeting different epitopes

  • Include isotype controls for monoclonal antibodies

Studies have demonstrated successful NARFL knockdown in A549 and H1299 cell lines, providing useful negative control materials for antibody validation .

How can NARFL antibodies help investigate mitochondrial dysfunction in cancer cells?

NARFL antibodies are valuable tools for studying mitochondrial dysfunction in cancer research through several methodological approaches:

• Correlation studies:

  • Use NARFL antibodies to quantify protein levels in cancer tissues

  • Correlate NARFL expression with mitochondrial integrity markers

  • Research has established that NARFL deficiency significantly correlates with:

    • Reduced mitochondrial Complex I activity

    • Decreased mtDNA copy numbers

    • Downregulated mRNA levels of mtND genes

    • Lower ATP levels

• Mechanistic investigations:

  • Combine NARFL immunostaining with mitochondrial markers

  • Evaluate changes in mitochondrial morphology and distribution in NARFL-deficient cells

  • Assess mitochondrial membrane potential using appropriate dyes in cells with differential NARFL expression

• Regulatory pathway analysis:

  • Study NARFL's relationship with key mitochondrial regulators

  • NARFL knockdown increases HIF-1α and DNMT1 protein levels, creating a HIF-1α-DNMT1 axis that mediates mitochondrial dysfunction

  • Use co-immunoprecipitation with NARFL antibodies to identify protein interaction partners

• Therapeutic response prediction:

  • NARFL deficiency increases drug resistance and cell migration in cancer cells

  • These effects can be reversed by silencing HIF-1α, suggesting a mechanistic relationship

  • NARFL antibodies can help stratify patient samples for potential therapeutic responsiveness

Research has demonstrated that NSCLC patients with NARFL deficiency have poor survival rates, highlighting the clinical relevance of NARFL detection in cancer samples .

What methodological approaches are needed to investigate NARFL's role in iron-sulfur cluster assembly?

To investigate NARFL's role in iron-sulfur cluster assembly, researchers should consider these methodological approaches:

• Protein interaction studies:

  • Use NARFL antibodies for co-immunoprecipitation to identify interaction partners

  • Combine with mass spectrometry to characterize NARFL-containing protein complexes

  • Analyze interactions with other known components of the cytosolic iron-sulfur cluster assembly pathway

• Enzyme activity assays:

  • Measure activities of iron-sulfur cluster-containing enzymes like cytosolic aconitase

  • Research has shown that NARFL knockout dramatically diminishes cytosolic aconitase activity

  • Correlate enzyme activities with NARFL protein levels quantified using antibodies

• Iron metabolism analysis:

  • Assess iron uptake, storage, and utilization in NARFL-deficient vs. normal cells

  • Combine NARFL antibody-based detection with iron sensors or stains

  • Monitor expression of iron regulatory proteins in relation to NARFL levels

• Redox homeostasis assessment:

  • Measure ROS levels in systems with variable NARFL expression

  • narfl deletion results in significantly elevated ROS levels (p < 0.001)

  • Evaluate antioxidant enzyme activities in relation to NARFL expression:

    • Cytosolic SOD activity is diminished in narfl-deficient models

    • GST activity is reduced in narfl-deficient models

    • Cytosolic GSH content is decreased in narfl mutants

• In vivo models:

  • Use NARFL antibodies to validate knockout efficiency in animal models

  • Research has documented that narfl knockout in zebrafish causes:

    • Larval lethality

    • Subintestinal vessel malformation

    • Digestive organ defects

These methodological approaches, combined with appropriate NARFL antibody applications, can provide comprehensive insights into NARFL's functional role in iron-sulfur cluster assembly.

How can researchers use NARFL antibodies to investigate HIF-1α signaling pathways?

NARFL antibodies can be instrumental in investigating HIF-1α signaling pathways through these methodological approaches:

• Expression correlation studies:

  • Quantify NARFL and HIF-1α protein levels in parallel using specific antibodies

  • NARFL knockdown increases HIF-1α protein levels, suggesting a regulatory relationship

  • In zebrafish models, narfl deficiency leads to increased expression of hif1ab and upregulated HIF-1α protein levels

• Mechanistic investigations:

  • Use NARFL antibodies in combination with HIF-1α target gene analysis

  • Perform ChIP assays to assess HIF-1α binding to target promoters in NARFL-deficient cells

  • Assess oxygen-dependent degradation of HIF-1α in relation to NARFL expression

• Signaling pathway integration:

  • NARFL deficiency activates a HIF-1α-DNMT1 axis that mediates mitochondrial dysfunction

  • Increased phosphorylated Akt (p-Akt) protein levels have been observed in narfl-deficient models

  • Combine NARFL antibody detection with analysis of these related pathways

• Pharmacological modulation studies:

  • The mitochondrial dysfunction caused by NARFL deficiency can be ameliorated by:

    • siHIF-1α (silencing HIF-1α)

    • DNMT1 inhibitors

  • Use NARFL antibodies to confirm protein levels while modulating these pathways

• Clinical correlation analysis:

  • Assess NARFL and HIF-1α expression patterns in patient samples

  • Correlate these patterns with clinical outcomes

  • NSCLC patients with NARFL deficiency show poor survival rates

These approaches can help elucidate the complex regulatory relationships between NARFL and hypoxia signaling pathways in normal physiology and disease states.

How should researchers interpret discrepancies between NARFL mRNA and protein expression?

When faced with discrepancies between NARFL mRNA and protein expression, researchers should consider several methodological and biological factors:

• Post-transcriptional regulation:

  • NARFL may be subject to microRNA regulation

  • RNA binding proteins might affect NARFL mRNA stability or translation efficiency

  • Investigate these possibilities by analyzing NARFL mRNA association with polysomes

• Protein stability considerations:

  • NARFL has three isoforms produced by alternative splicing

  • Different isoforms may have distinct stability profiles

  • Use pulse-chase experiments with NARFL antibodies to assess protein half-life

• Technical validation approaches:

  • Verify mRNA measurements using multiple primer sets targeting different exons

  • Confirm protein measurements using different NARFL antibodies targeting distinct epitopes

  • The observed molecular weight of NARFL (26-28 kDa) differs from calculated (53 kDa) , suggesting potential processing that might affect detection

• Experimental design considerations:

  • Ensure temporal alignment between mRNA and protein measurements

  • Consider that NARFL mRNA changes may precede protein changes or vice versa

  • Design time-course experiments to capture the relationship accurately

• Cellular context factors:

  • Iron availability might affect NARFL protein stability without altering mRNA

  • Hypoxic conditions could differentially affect NARFL mRNA versus protein

  • NARFL's role in iron-sulfur cluster assembly suggests it may be regulated by cellular iron status

When studying NARFL in relation to cancer, note that NSCLC patients with NARFL deficiency show poor survival rates , highlighting the importance of accurate protein quantification alongside mRNA analysis.

What controls should be included when using NARFL antibodies in experimental designs?

A robust experimental design using NARFL antibodies should include the following controls:

• Positive controls:

  • Cell lines with confirmed NARFL expression:

    • HeLa cells

    • HEK-293 cells

  • Tissues with validated NARFL staining:

    • Human colon tissue

• Negative controls:

  • NARFL knockdown samples:

    • A549 and H1299 cell lines with NARFL knockdown have been validated

    • Include siRNA NARFL-treated samples

  • No primary antibody controls

  • Isotype controls for monoclonal antibodies

• Specificity controls:

  • Pre-absorption with immunogen when available

  • Competition assays with recombinant NARFL protein

  • Compare results from multiple antibodies targeting different NARFL epitopes

• Technical controls:

  • Loading controls for Western blots (β-actin, GAPDH, etc.)

  • Housekeeping genes for normalization in qPCR

  • For IHC/IF, include tissue sections known to express or lack NARFL

• Experimental validation controls:

  • For functional studies on NARFL's role in Fe-S protein biogenesis:

    • Measure cytosolic aconitase activity (diminished in NARFL knockout)

  • For oxidative stress studies:

    • Include ROS measurements (elevated in NARFL-deficient models)

    • Assess antioxidant enzyme activities (SOD, GST)

• Rescue controls:

  • Re-express NARFL in knockdown/knockout systems

  • Verify phenotype rescue with appropriate NARFL antibody detection

These comprehensive controls will enhance the reliability and interpretability of experiments using NARFL antibodies.

How can researchers correlate NARFL levels with clinical outcomes in cancer patients?

To effectively correlate NARFL levels with clinical outcomes in cancer patients, researchers should implement these methodological approaches:

Research has demonstrated that NARFL deficiency causes dysregulation of energy metabolism in lung cancer cells via a HIF-1α–DNMT1 axis, which promotes drug resistance and cell migration . These mechanistic insights provide a biological foundation for clinical correlative studies.

How might NARFL antibodies facilitate research on redox homeostasis in disease models?

NARFL antibodies can advance research on redox homeostasis in disease models through several methodological approaches:

• Oxidative stress analysis:

  • Correlate NARFL protein levels with ROS measurements

  • narfl deletion results in significantly elevated ROS levels (p < 0.001)

  • This effect can be downregulated by N-acetylcysteine (NAC) treatment

  • Use NARFL antibodies to confirm protein expression levels in these experimental systems

• Antioxidant system evaluation:

  • Assess the relationship between NARFL expression and antioxidant enzyme activities

  • In narfl-deficient models:

    • Cytosolic SOD activity is diminished

    • GST activity is reduced

    • Cytosolic GSH content is decreased

• Subcellular localization studies:

  • Use NARFL antibodies for immunofluorescence to track protein localization under oxidative stress

  • Employ subcellular fractionation followed by Western blotting to quantify NARFL distribution

  • Correlate localization patterns with markers of oxidative damage

• Therapeutic intervention assessment:

  • Evaluate how antioxidant treatments affect NARFL expression and function

  • Investigate whether NARFL overexpression can mitigate oxidative stress in disease models

  • Use NARFL antibodies to monitor protein levels during therapeutic interventions

• Pathological applications:

  • In cancer research:

    • NARFL deficiency promotes mitochondrial dysfunction in lung cancer cells

    • This leads to increased drug resistance and cell migration

  • In vascular development:

    • narfl deletion in zebrafish causes subintestinal vessel malformation

    • NARFL is a causative gene for diffused pulmonary arteriovenous malformations (dPAVMs)

These approaches can help elucidate NARFL's role in maintaining redox homeostasis across different physiological and pathological contexts.

What novel methodologies are emerging for studying NARFL-protein interactions?

Emerging methodologies for studying NARFL-protein interactions include:

• Proximity labeling approaches:

  • BioID or TurboID fusion with NARFL to identify proximal proteins

  • APEX2-based proximity labeling in living cells

  • These methods can reveal transient or weak interactions missed by traditional co-IP

  • Validate findings using conventional co-IP with NARFL antibodies

• Advanced microscopy techniques:

  • Super-resolution microscopy to visualize NARFL localization at nanoscale resolution

  • FRET/FLIM assays to detect direct protein-protein interactions

  • Live-cell imaging with fluorescently-tagged NARFL to track dynamic interactions

  • These approaches can benefit from validation with fixed-cell immunofluorescence using NARFL antibodies

• Mass spectrometry-based interactomics:

  • Crosslinking mass spectrometry (XL-MS) to map interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to detect conformational changes

  • Immunoprecipitation with NARFL antibodies followed by quantitative proteomics

  • These techniques can reveal detailed molecular mechanisms of NARFL function

• Protein-protein interaction screening platforms:

  • Mammalian two-hybrid systems adapted for NARFL studies

  • Protein complementation assays using split fluorescent or luminescent reporters

  • High-throughput yeast two-hybrid screens with NARFL as bait

  • Confirm interactions using orthogonal methods with NARFL antibodies

• Advanced structural biology applications:

  • Cryo-EM studies of NARFL-containing complexes

  • Integrative structural modeling combining multiple data sources

  • NARFL antibody fragments can potentially be used to stabilize complexes for structural studies

These emerging methodologies, when combined with validated NARFL antibodies, can provide unprecedented insights into NARFL's functional interactions in iron-sulfur cluster assembly and beyond.