NFYA2 Antibody

What is the molecular structure of NF2/Merlin protein and how does it affect antibody selection?

NF2/Merlin is a 70-75 kDa tumor suppressor protein belonging to the ERM (ezrin, radixin, moesin) family. Human NF2 consists of 595 amino acids with three distinct structural regions: an N-terminal FERM domain (amino acids 1-302), an α-helical rod central region (amino acids 303-478), and a unique carboxy-terminal domain (amino acids 479-595) . When selecting antibodies, researchers should consider the structural complexity of NF2/Merlin and choose antibodies that target stable epitopes. For optimal experimental outcomes, antibodies recognizing conserved regions are preferred for cross-species studies, while those targeting the unique C-terminal domain can provide higher specificity for human samples.

What applications are NF2/Merlin antibodies validated for?

NF2/Merlin antibodies have been validated for multiple research applications with specific optimization parameters:

These applications enable researchers to investigate NF2/Merlin expression, localization, and interactions across various experimental systems . It's important to note that optimal dilutions should be determined by each laboratory for specific applications and samples.

How can I validate the specificity of my NF2/Merlin antibody?

A methodological approach to validate NF2/Merlin antibody specificity should include:

• Positive control selection: Use cell lines with documented NF2/Merlin expression such as HeLa, HepG2, or MCF-7 cells, which consistently show bands at 70 kDa in Western blots .

• Knockout/knockdown validation: Compare antibody reactivity between wild-type samples and those with NF2 gene knockdown or knockout. Published literature using this approach can provide reference data .

• Multiple antibody comparison: Use antibodies targeting different epitopes of NF2/Merlin to confirm consistent detection patterns.

• Peptide competition assay: Pre-incubate antibody with purified NF2/Merlin protein or peptide to demonstrate signal reduction in subsequent applications.

These validation steps ensure experimental results accurately reflect NF2/Merlin biology rather than non-specific interactions.

What are the critical considerations when designing experiments to study NF2/Merlin phosphorylation states?

NF2/Merlin functionality is significantly regulated by phosphorylation, particularly at Ser518. When investigating phosphorylation states:

• Select phospho-specific antibodies: Use antibodies specifically targeting phosphorylated residues, such as phospho-NF2 (Ser518) antibodies .

• Include appropriate controls: Parallel analysis with phospho-specific and total NF2/Merlin antibodies provides accurate assessment of phosphorylation levels relative to total protein.

• Phosphatase treatment controls: Include samples treated with phosphatases to confirm phospho-antibody specificity.

• Consider cell confluence effects: NF2/Merlin phosphorylation is influenced by cell density, with confluence requiring absence of phosphorylation at Ser516 and Ser10 . Design experiments with consistent cell densities.

• Sample preparation optimization: Use phosphatase inhibitors during cell lysis to preserve in vivo phosphorylation states.

This approach enables accurate characterization of NF2/Merlin's phosphorylation-dependent functions in cell adhesion, proliferation regulation, and tumor suppression pathways.

How can I distinguish between NF2/Merlin splice variants in my experiments?

NF2/Merlin exists in multiple splice variants that affect protein function. A systematic approach to distinguish these variants includes:

• Selection of domain-specific antibodies: Choose antibodies targeting regions present or absent in specific splice variants. The search results indicate multiple potential splice variants, including eight isoforms showing alternate start sites coupled with an 11 amino acid substitution for positions 579-595 .

• Higher resolution gel systems: Use 8-10% acrylamide gels with extended run times to separate closely migrating isoforms.

• 2D gel electrophoresis: Combined with Western blotting, this approach can separate isoforms by both molecular weight and isoelectric point.

• Complementary RNA analysis: Pair protein detection with RT-PCR using isoform-specific primers to confirm the presence of specific splice variants.

• Mass spectrometry validation: For definitive identification, immunoprecipitate NF2/Merlin and perform mass spectrometry analysis of tryptic fragments.

This multi-faceted approach provides a comprehensive characterization of NF2/Merlin isoforms in experimental systems, crucial for understanding their differential functions.

What experimental approaches are most effective for studying NF2/Merlin's role in signaling pathways?

NF2/Merlin participates in multiple signaling cascades, including Hippo, mTOR, Wnt/β-catenin, TGF-β, and other pathways critical for cell growth regulation . Effective experimental strategies include:

• Co-immunoprecipitation (Co-IP): Use NF2/Merlin antibodies (0.5-4.0 μg per 1.0-3.0 mg of protein lysate) to pull down interaction partners, followed by immunoblotting for pathway components .

• Proximity ligation assays: Visualize and quantify protein-protein interactions between NF2/Merlin and pathway components with subcellular resolution.

• Pathway reporter assays: Measure downstream transcriptional activity (e.g., TEAD reporters for Hippo pathway) in wild-type versus NF2-deficient cells.

• Phosphorylation cascade analysis: Monitor phosphorylation states of downstream effectors (e.g., YAP/TAZ in Hippo pathway) using phospho-specific antibodies.

• Drug-target interaction studies: Combine NF2/Merlin detection with inhibitors of mTOR, HDAC, or VEGF to characterize pathway crosstalk.

These approaches facilitate mechanistic insights into how NF2/Merlin functions as a tumor suppressor through regulation of diverse signaling networks .

How can I resolve inconsistent Western blot results with NF2/Merlin antibodies?

Inconsistent NF2/Merlin detection in Western blots can be addressed through systematic optimization:

• Sample preparation refinement:

  • Use stringent lysis buffers containing SDS or strong detergents to fully solubilize membrane-associated NF2/Merlin

  • Maintain samples at 4°C and include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors as phosphorylation affects antibody recognition

• Transfer optimization:

  • For the 70 kDa NF2/Merlin protein, use 0.45 μm PVDF membranes

  • Extend transfer time or use wet transfer systems for more complete protein transfer

• Blocking and antibody incubation:

  • Test alternative blocking agents (5% milk vs. BSA) as NF2/Merlin detection can be sensitive to blocking conditions

  • Optimize primary antibody dilution within the recommended range (1:2000-1:16000)

  • Extend incubation time to overnight at 4°C for better signal-to-noise ratio

• Detection system selection:

  • Use enhanced chemiluminescence (ECL) with higher sensitivity for low expression samples

  • Consider fluorescent secondary antibodies for more quantitative analysis

• Control inclusion:

  • Run validated positive controls like HeLa or Jurkat cell lysates in parallel

  • Include loading controls that separate well from the 70 kDa region

These methodological refinements significantly improve consistency and reproducibility in NF2/Merlin Western blot experiments.

What strategies can overcome challenges in immunohistochemical detection of NF2/Merlin?

Immunohistochemical detection of NF2/Merlin presents several technical challenges that can be addressed through:

• Optimized antigen retrieval:

  • Use TE buffer at pH 9.0 as the primary method, with citrate buffer pH 6.0 as an alternative

  • Determine optimal retrieval time (typically 15-20 minutes) for each tissue type

  • For formalin-fixed tissues, extend retrieval time to ensure complete epitope exposure

• Signal amplification approaches:

  • Implement tyramide signal amplification for low-abundance detection

  • Use polymer-based detection systems for improved sensitivity

  • Consider biotin-free detection systems to reduce background in tissues with endogenous biotin

• Antibody optimization:

  • Test multiple antibody concentrations within the recommended range (1:750-1:3000)

  • Extend primary antibody incubation to overnight at 4°C

  • Validate antibody performance in tissues with known NF2/Merlin expression patterns

• Background reduction:

  • Include additional blocking steps with normal serum matching the secondary antibody species

  • Use tissue-specific blocking agents for high-background samples

  • Implement avidin/biotin blocking for endogenous biotin-containing tissues

• Controls and validation:

  • Include positive control tissues with known NF2/Merlin expression (e.g., human meningioma tissue)

  • Use tissues from NF2 knockout models as negative controls

These approaches significantly improve detection specificity and signal-to-noise ratio in NF2/Merlin immunohistochemistry experiments.

How can NF2/Merlin antibodies be effectively utilized in studying tumor metabolism?

Recent research has established connections between NF2/Merlin and cancer metabolic reprogramming . Researchers can leverage NF2/Merlin antibodies to investigate these metabolic phenotypes through:

• Co-localization with metabolic enzymes:

  • Use dual immunofluorescence with NF2/Merlin antibodies (1:200-1:800 dilution) and antibodies against key metabolic enzymes

  • Analyze subcellular co-localization in normal versus tumor tissues

• Metabolic pathway investigation:

  • Combine NF2/Merlin immunoprecipitation with metabolic enzyme activity assays

  • Correlate NF2/Merlin expression/phosphorylation with metabolic enzyme levels across tumor specimens

• Tumor microenvironment analysis:

  • Apply multiplex immunohistochemistry to simultaneously detect NF2/Merlin and metabolic markers

  • Quantify spatial relationships between NF2/Merlin expression and metabolic zonation in tumors

• Therapeutic response markers:

  • Monitor NF2/Merlin levels and phosphorylation state as biomarkers during treatment with metabolic inhibitors

  • Correlate metabolic drug sensitivity with NF2/Merlin status using immunoblotting

This approach provides mechanistic insights into how NF2/Merlin deficiency drives metabolic alterations that support tumorigenesis, potentially identifying new therapeutic vulnerabilities .

What methodological approaches can reveal the role of NF2/Merlin in embryonic development?

NF2/Merlin plays critical roles in embryogenesis, with NF2 deficiency causing severe developmental defects and embryonic lethality . To investigate these developmental functions:

• Temporal expression profiling:

  • Use NF2/Merlin antibodies in Western blots of embryonic tissues at different developmental stages

  • Quantify expression changes across developmental timepoints

• Spatial localization analysis:

  • Apply immunohistochemistry (1:750-1:3000 dilution) to embryonic tissue sections

  • Map NF2/Merlin distribution in developing organs and tissues

• Co-expression with developmental markers:

  • Perform dual immunofluorescence to correlate NF2/Merlin with tissue-specific developmental markers

  • Analyze co-localization patterns during different developmental stages

• Developmental signaling integration:

  • Combine NF2/Merlin detection with markers of Hippo, Wnt/β-catenin, and other developmental pathways

  • Characterize the relationship between NF2/Merlin and developmental signaling networks

• Genetic model validation:

  • Use NF2/Merlin antibodies to confirm knockdown/knockout efficiency in developmental models

  • Compare protein expression with developmental phenotypes

These approaches provide mechanistic insights into how NF2/Merlin regulates critical developmental processes and how its dysfunction leads to developmental abnormalities.

How can I design experiments to study the interplay between NF2/Merlin and the immune microenvironment?

Emerging research suggests connections between NF2/Merlin and tumor immunity . To investigate this relationship:

• Immune cell co-localization studies:

  • Perform multiplex immunofluorescence with NF2/Merlin antibodies and immune cell markers

  • Quantify spatial relationships between NF2/Merlin-expressing cells and immune populations

• Immune checkpoint correlation analysis:

  • Use sequential immunohistochemistry to detect NF2/Merlin and immune checkpoint molecules

  • Analyze correlation patterns across tumor specimens

• Cytokine response experiments:

  • Measure changes in NF2/Merlin expression/phosphorylation after cytokine treatment

  • Use Western blotting to quantify NF2/Merlin protein levels in response to immune stimulation

• Immune signaling pathway integration:

  • Perform co-immunoprecipitation to identify interactions between NF2/Merlin and immune signaling components

  • Study how NF2/Merlin status affects downstream immune signaling cascades

• Therapeutic response biomarkers:

  • Monitor NF2/Merlin as a potential biomarker during immunotherapy treatment

  • Correlate treatment responses with NF2/Merlin expression patterns

This experimental framework reveals how NF2/Merlin deficiency shapes the tumor immune microenvironment, potentially informing immunotherapeutic approaches for NF2-mutated tumors .

What emerging technologies show promise for advancing NF2/Merlin antibody-based research?

Several cutting-edge technologies are poised to transform NF2/Merlin antibody applications:

• Single-cell proteomics:

  • Apply NF2/Merlin antibodies in mass cytometry (CyTOF) for single-cell protein quantification

  • Integrate with transcriptomic data for multi-omic analysis of NF2/Merlin biology

• Super-resolution microscopy:

  • Utilize fluorescently-labeled NF2/Merlin antibodies in STORM or PALM microscopy

  • Achieve nanoscale resolution of NF2/Merlin localization and interactions

• Proximity-dependent labeling:

  • Combine NF2/Merlin antibodies with BioID or APEX techniques

  • Map the dynamic NF2/Merlin interactome in different cellular contexts

• Spatial transcriptomics integration:

  • Correlate NF2/Merlin protein localization with spatial gene expression patterns

  • Develop comprehensive maps of NF2/Merlin function across tissue microenvironments

• Computational antibody improvement:

  • Apply artificial intelligence for epitope optimization

  • Design next-generation NF2/Merlin antibodies with enhanced specificity and sensitivity