NUP98 Monoclonal Antibody

What is NUP98 and why is it important in cellular biology?

NUP98 is an essential nucleoporin and component of the nuclear pore complex (NPC) that plays critical roles in nuclear functions including transcriptional regulation and nucleocytoplasmic transport. It is involved in nuclear pore complex assembly and maintenance, working together with NUP96 to facilitate bidirectional transport across the NPC . NUP98 may anchor other nucleoporins such as NUP153 and TPR to the NPC. Additionally, in cooperation with DHX9, NUP98 plays an important role in transcription and alternative splicing activation of a subset of genes, as well as in the localization of DHX9 in discrete intranuclear foci called GLFG-bodies . These diverse functions make NUP98 a critical protein for studying nuclear transport mechanisms, gene regulation, and various disease states including certain leukemias.

What are the key structural features of NUP98 that monoclonal antibodies typically target?

The most significant structural feature of NUP98 targeted by monoclonal antibodies is the Gly-Leu-Phe-Gly (GLFG) sequence that appears repetitively in the N-terminal region of the protein . This sequence is well-conserved among NUP98 proteins from a wide variety of species including humans, yeasts, and ciliates such as Tetrahymena thermophila, making it an ideal target for antibodies with broad species reactivity . Specific epitopes within this region include the FGxxN motif (where x represents any amino acid) recognized by the 13C2 monoclonal antibody and the GLF motif recognized by the 21A10 monoclonal antibody . Commercial antibodies like the 2H10 rat monoclonal antibody are often raised against recombinant fragment proteins within the human NUP98 amino acid range 1-500, which contains these conserved GLFG repeats .

What applications are NUP98 monoclonal antibodies most commonly used for?

NUP98 monoclonal antibodies are predominantly used in several key applications:

• Western Blot (WB): For detecting NUP98 protein in cell or tissue lysates, typically appearing at approximately 98 kDa .

• Immunocytochemistry/Immunofluorescence (ICC/IF): For visualizing the subcellular localization of NUP98, particularly at the nuclear periphery where nuclear pores are located .

• Nuclear Pore Complex (NPC) studies: As markers for studying NPC assembly, structure, and function .

• Cross-species studies: Some NUP98 antibodies that recognize conserved epitopes can be used across multiple species, from yeast to humans .

• Pathological studies: For investigating NUP98-related hematopoietic malignancies and leukemias .

The choice of which antibody to use depends on the specific application and species being studied, as different antibodies show varying levels of effectiveness across applications.

How can I verify the specificity of a NUP98 monoclonal antibody in my experimental system?

To verify the specificity of a NUP98 monoclonal antibody in your experimental system, implement the following methodological approach:

• Positive control validation: Use cell lines known to express NUP98 (most mammalian cell lines express it) to confirm expected staining pattern and molecular weight .

• Knockout/knockdown validation: Compare antibody reactivity in wild-type cells versus NUP98 knockout or knockdown cells. A specific antibody will show significantly reduced or absent signal in the knockout/knockdown samples .

• Epitope competition assay: Pre-incubate the antibody with purified peptide containing the target epitope before application to your sample. Specific binding should be blocked by the competing peptide .

• Immunofluorescence pattern analysis: Confirm that the staining pattern shows characteristic nuclear rim localization typical of nuclear pore complex proteins .

• Molecular weight verification: In Western blots, NUP98 should appear at approximately 98 kDa. For tagged versions (like GFP-NUP98), the band should shift by the expected molecular weight of the tag (e.g., approximately 28 kDa for GFP) .

• Cross-reactivity assessment: If working with non-human samples, verify whether the antibody recognizes the conserved GLFG epitopes across species by testing on multiple species samples if available .

What are the optimal sample preparation methods for using NUP98 antibodies in immunofluorescence?

For optimal immunofluorescence results with NUP98 antibodies, the following detailed protocol is recommended:

• Fixation:

  • For adherent cells: Fix with 4% paraformaldehyde in PBS for 15 minutes at room temperature, followed by permeabilization with 0.5% Triton X-100 for 10 minutes .

  • For suspension cells: Cytospin cells onto slides before following the same fixation procedure.

• Blocking:

  • Block with 1% BSA in PBS for 2 hours at room temperature to minimize non-specific binding .

• Primary antibody incubation:

  • Dilute NUP98 monoclonal antibodies to approximately 0.5 μg/mL in PBS with 1% BSA.

  • Incubate samples overnight at 4°C for optimal binding .

• Secondary antibody treatment:

  • Use appropriate species-specific secondary antibodies (anti-mouse IgG for 13C2 and 21A10; anti-rat IgG for 2H10) conjugated to fluorophores at approximately 4 μg/mL.

  • Incubate for 1-2 hours at room temperature .

• Nuclear counterstaining:

  • Counterstain nuclei with DAPI (4',6-diamidino-2-phenylindole) to provide context for NUP98 localization .

• Mounting and imaging:

  • Mount slides with anti-fade mounting medium.

  • Image using high-resolution confocal microscopy with appropriate oil-immersion objectives (NA≥1.3) to clearly visualize the nuclear envelope and pore complexes .

Between each step, wash samples three times with PBS to remove unbound reagents and reduce background signal .

What experimental controls should be included when using NUP98 antibodies for Western blotting?

When using NUP98 antibodies for Western blotting, the following comprehensive set of controls should be included:

For optimal Western blot results, use adequate protein amounts (typically 20-40 μg of total protein per lane), ensure complete protein transfer, and optimize antibody dilutions (typically starting at 1:1000 for NUP98 monoclonal antibodies) .

How should I optimize antibody concentrations for different experimental applications?

Optimizing antibody concentrations is critical for achieving specific signals while minimizing background. Here's a methodological approach for different applications:

For Western Blotting:

• Initial titration: Start with a dilution series (1:500, 1:1000, 1:2000, 1:5000) of the NUP98 antibody.

• Signal evaluation: Select the concentration that provides clear specific bands at 98 kDa with minimal background.

• Fine-tuning: If background remains high, adjust blocking conditions (5% milk vs. 5% BSA) or increase washing stringency.

• Secondary antibody adjustment: Typically use secondary antibodies at 1:5000-1:10,000 dilution to reduce background.

For Immunofluorescence:

• Initial screening: Test a concentration gradient (0.1, 0.5, 1.0, 5.0 μg/mL) of the NUP98 antibody.

• Pattern analysis: Look for distinct nuclear rim staining characteristic of nuclear pore proteins.

• Signal-to-noise optimization: Select the lowest concentration that gives clear nuclear rim staining with minimal cytoplasmic background.

• Secondary antibody optimization: Typically use fluorophore-conjugated secondary antibodies at 2-4 μg/mL .

For Flow Cytometry:

• Initial concentration range: Test antibody at multiple concentrations (1-10 μg/mL).

• Titration analysis: Plot median fluorescence intensity against antibody concentration to identify the saturation point.

• Comparison with isotype control: Ensure specific staining compared to isotype control at the same concentration.

For each new lot of antibody, perform a re-optimization as antibody activity can vary between lots. Document the optimal conditions determined for future reference and reproducibility.

How can NUP98 monoclonal antibodies be used to study NUP98 fusion proteins in leukemia research?

NUP98 monoclonal antibodies are valuable tools for studying NUP98 fusion proteins in leukemia research through several sophisticated approaches:

• Detection and characterization of fusion proteins:

  • NUP98 antibodies that recognize epitopes in the N-terminal GLFG repeat region can detect various NUP98 fusion proteins, as this region is typically retained in the fusion events .

  • Western blotting with these antibodies can identify abnormal molecular weight bands representing fusion proteins in patient samples or experimental models.

• Subcellular localization studies:

  • Immunofluorescence microscopy using NUP98 antibodies can reveal altered subcellular localization patterns of fusion proteins compared to wild-type NUP98 .

  • This is particularly important as NUP98 fusion proteins often show aberrant localization away from the nuclear pore complex, correlating with their pathological functions.

• Co-immunoprecipitation experiments:

  • NUP98 antibodies can be used to pull down NUP98 fusion proteins and identify novel interaction partners that contribute to leukemogenic transformation.

  • Comparing interactomes between wild-type NUP98 and fusion variants can reveal mechanistic insights into pathogenesis.

• Chromatin immunoprecipitation (ChIP) assays:

  • As many NUP98 fusion proteins function as aberrant transcriptional regulators, NUP98 antibodies can be employed in ChIP experiments to map genomic binding sites.

  • This approach can identify target genes dysregulated by NUP98 fusion proteins in leukemia cells.

• Patient sample analysis:

  • NUP98 antibodies can be used to screen patient samples for abnormal expression or localization of NUP98, potentially identifying cases with NUP98 rearrangements even before genetic confirmation .

When selecting antibodies for these applications, researchers should verify whether the epitope recognized by the antibody is retained in the specific fusion protein being studied, as this varies depending on the fusion partner and breakpoint location.

What are the key differences between available NUP98 monoclonal antibodies, and how do they affect experimental outcomes?

Different NUP98 monoclonal antibodies target distinct epitopes and demonstrate varying performance characteristics that significantly impact experimental outcomes:

These differences affect experimental outcomes in several ways:

• Epitope availability: In certain experimental conditions or fixation methods, specific epitopes may be masked or denatured, affecting antibody binding.

• Cross-reactivity profiles: Antibodies recognizing GLF motifs (like 21A10) may detect other nucleoporins containing similar motifs, potentially complicating interpretation unless proper controls are employed .

• Species-specific studies: For evolutionary or comparative studies, antibodies with broad species reactivity (like 13C2 and 21A10) offer advantages in detecting NUP98 homologs across diverse organisms .

• Fusion protein detection: Depending on the fusion breakpoint, certain epitopes may be lost in NUP98 fusion proteins, necessitating careful antibody selection for leukemia research.

• Application compatibility: Some antibodies perform significantly better in certain applications (e.g., 13C2 for Western blot; 21A10 for immunofluorescence), making it important to select the appropriate antibody for each specific experimental context .

How can NUP98 antibodies be used to study the dynamics of nuclear pore complex assembly?

NUP98 monoclonal antibodies can be powerful tools for investigating the complex dynamics of nuclear pore complex (NPC) assembly through several sophisticated methodological approaches:

• Live-cell imaging with fluorescently tagged antibody fragments:

  • Antibody-derived Fab fragments labeled with fluorescent dyes can track NUP98 incorporation into assembling NPCs in living cells.

  • This approach requires careful validation that the antibody binding doesn't interfere with NUP98 function or localization.

• Pulse-chase immunoprecipitation studies:

  • NUP98 antibodies can be used to pull down newly synthesized NUP98 and its interaction partners at different time points during NPC assembly.

  • This reveals the temporal sequence of protein recruitment and complex formation.

• Cell cycle synchronized immunofluorescence analysis:

  • By synchronizing cells at different cell cycle stages and performing quantitative immunofluorescence with NUP98 antibodies, researchers can map the incorporation of NUP98 into NPCs during post-mitotic nuclear envelope reassembly .

  • Co-staining with other nucleoporin antibodies can establish the order of recruitment.

• Correlative light and electron microscopy (CLEM):

  • NUP98 antibodies conjugated to both fluorescent tags and electron-dense markers enable visualization of NUP98 localization during NPC assembly at both light microscopy and ultrastructural levels.

  • This approach can reveal structural intermediates during NPC biogenesis.

• Study of annulate lamellae:

  • NUP98-deficient cells show approximately 5-fold increase in cytoplasmic annulate lamellae compared to control cells .

  • NUP98 antibodies can be used to study the relationship between these cytoplasmic pore complex-containing structures and nuclear envelope-embedded NPCs.

• In vitro reconstitution assays:

  • Using cell-free systems, researchers can add labeled NUP98 antibodies to track the incorporation of NUP98 into assembling NPCs in real-time.

  • This approach allows for manipulation of assembly conditions and identification of rate-limiting steps.

These methodologies provide complementary insights into the complex process of NPC assembly and the specific role of NUP98 in this essential cellular process.

Why might I observe inconsistent staining patterns with NUP98 antibodies in immunofluorescence experiments?

Inconsistent staining patterns with NUP98 antibodies can result from several technical and biological factors that should be systematically investigated:

• Fixation and permeabilization variables:

  • Different fixation methods (paraformaldehyde vs. methanol) affect epitope accessibility differently.

  • Over-fixation can mask epitopes while insufficient permeabilization may prevent antibody access to nuclear pore complexes .

  • Solution: Optimize fixation time (typically 10-15 minutes with 4% PFA) and test different permeabilization agents (0.1-0.5% Triton X-100, 0.1-0.2% Saponin).

• Cell cycle stage variations:

  • NUP98 distribution changes during the cell cycle, particularly during mitosis when the nuclear envelope breaks down.

  • Solution: Synchronize cells or co-stain with cell cycle markers to correlate staining patterns with cell cycle stages.

• Antibody-specific factors:

  • Different antibody clones recognize distinct epitopes with varying accessibility.

  • For example, 21A10 (recognizing GLF) generally performs better in immunofluorescence than 13C2 (recognizing FGxxN) .

  • Solution: Test multiple NUP98 antibody clones and select the most consistent performer for your experimental system.

• Species and isoform differences:

  • NUP98 homologs across species have variable epitope conservation.

  • Solution: Verify whether your antibody has been validated for your species of interest. For example, commercial antibody 2H10 works well with mammalian cells but poorly with T. thermophila .

• Sample preparation artifacts:

  • Nuclear envelope damage during processing can lead to irregular staining.

  • Solution: Use gentle handling during all preparation steps and consider alternative sample preparation methods.

• Technical inconsistencies:

  • Variability in antibody concentration, incubation time, or washing protocols.

  • Solution: Standardize all protocol steps, prepare fresh dilutions of antibodies, and maintain consistent incubation times and temperatures .

• Biological heterogeneity:

  • NUP98 expression or localization may naturally vary between cells in your population.

  • Solution: Quantify staining patterns across a large number of cells and consider single-cell approaches to address heterogeneity.

How can I differentiate between specific and non-specific binding when using NUP98 antibodies in Western blot analysis?

Differentiating between specific and non-specific binding in Western blot analysis using NUP98 antibodies requires a systematic analytical approach:

• Molecular weight verification:

  • Specific NUP98 signal should appear at approximately 98 kDa .

  • Additional bands may represent:

    • NUP98 fusion proteins (in disease samples)

    • Degradation products (lower MW)

    • Post-translationally modified forms

    • Non-specific binding

• Critical control experiments:

  • Knockout/knockdown validation: Compare wild-type to NUP98-depleted samples; specific bands should be reduced or absent in depleted samples .

  • Overexpression control: Cells expressing tagged NUP98 should show an additional band at the expected shifted molecular weight (e.g., GFP-NUP98 at ~125-130 kDa) .

  • Peptide competition: Pre-incubation of antibody with its target peptide should eliminate specific bands but not non-specific ones.

• Comparative antibody analysis:

  • Use multiple antibodies targeting different NUP98 epitopes (e.g., 13C2, 21A10, 2H10) .

  • Specific bands should be detected by multiple antibodies while non-specific bands typically vary between antibodies.

• Optimization of blocking and washing:

  • Non-specific binding often appears as multiple bands or high background.

  • Test different blocking agents (5% milk vs. 5% BSA) and more stringent washing conditions.

  • Consider adding 0.1-0.5% Tween-20 or 0.1% SDS to washing buffers to reduce non-specific binding.

• Signal intensity analysis:

  • Specific signals typically show consistent intensity ratios between samples when normalized to loading controls.

  • Non-specific bands often show random variation between replicates or samples.

• Gradient gel analysis:

  • Run samples on gradient gels (4-12% or 4-20%) to better separate proteins in the 90-120 kDa range.

  • This approach can help distinguish closely migrating specific and non-specific bands.

• Tissue/cell type specificity patterns:

  • NUP98 is ubiquitously expressed in nucleated cells.

  • If a band appears only in certain tissues where NUP98 shouldn't be differentially expressed, it's likely non-specific.

How should data from NUP98 antibody experiments be interpreted in the context of nucleoporin function and disease states?

Interpreting data from NUP98 antibody experiments in functional and disease contexts requires integration of multiple experimental findings and consideration of NUP98's diverse roles:

• Nuclear pore complex (NPC) assembly and maintenance:

  • NUP98 is essential for proper NPC assembly and bidirectional transport .

  • Altered NUP98 localization or expression may indicate NPC dysfunction.

  • Interpretation framework: Changes in NUP98 staining patterns at the nuclear envelope often correlate with altered nucleocytoplasmic transport efficiency. Quantify nuclear rim staining intensity and pattern uniformity as indicators of NPC integrity.

• Transcriptional regulation:

  • NUP98 cooperates with DHX9 in transcription and alternative splicing activation .

  • Interpretation framework: Intranuclear NUP98 foci (distinct from the nuclear envelope) may indicate sites of transcriptional activity. Correlate these with nascent RNA synthesis markers and transcription factor localization.

• Leukemia-associated NUP98 fusions:

  • NUP98 is involved in chromosomal translocations in hematological malignancies .

  • Interpretation approach: When analyzing patient samples:

    • Abnormal molecular weight bands in Western blots may indicate fusion proteins

    • Altered subcellular localization (nuclear body localization instead of nuclear rim) suggests fusion protein activity

    • Compare with known leukemia cell lines harboring NUP98 fusions as positive controls

• Developmental contexts:

  • NUP98 is essential for mouse gastrulation but dispensable for basal cell growth .

  • Interpretation framework: Developmental phenotypes may result from altered gene expression patterns rather than direct nucleocytoplasmic transport defects. Correlate NUP98 antibody staining with markers of cellular differentiation.

• Viral interactions:

  • NUP98 interacts with HIV-1 capsid and nucleocapsid proteins, potentially promoting viral integration .

  • Interpretation approach: In infection studies, co-localization between NUP98 and viral proteins should be quantified and compared to other nucleoporins to establish specificity.

• Annulate lamellae formation:

  • NUP98-deficient cells show 5-fold increase in cytoplasmic annulate lamellae .

  • Interpretation framework: Excessive cytoplasmic NUP98 staining in punctate structures may indicate annulate lamellae formation, which often occurs in rapidly dividing cells and certain disease states.

• Cross-species analysis:

  • NUP98's GLFG repeats are conserved across diverse species .

  • Interpretation approach: When using antibodies across species, confirm epitope conservation through sequence analysis and validate with appropriate controls for each species.

How are NUP98 monoclonal antibodies being used in current research on nuclear transport mechanisms?

Current research is employing NUP98 monoclonal antibodies in increasingly sophisticated ways to elucidate nuclear transport mechanisms:

• Super-resolution microscopy studies:

  • NUP98 antibodies are being used with techniques like STORM, PALM, and structured illumination microscopy to visualize the precise arrangement of NUP98 within the nuclear pore complex at nanometer resolution.

  • These approaches have revealed that NUP98 forms a dynamic meshwork at the nuclear pore that can regulate transport selectivity .

• Transport kinetics analyses:

  • By combining NUP98 immunostaining with real-time cargo tracking, researchers are correlating NUP98 distribution with transport efficiency.

  • This has revealed that NUP98 and NUP96 are involved in bidirectional transport across the NPC, with different domains serving distinct functions .

• Interactome mapping:

  • Immunoprecipitation with NUP98 antibodies followed by mass spectrometry is being used to comprehensively map the protein interaction network of NUP98.

  • Recent studies have identified interactions with specific transport receptors and their cargoes, suggesting direct regulatory roles in transport beyond structural functions .

• Phase separation studies:

  • NUP98's FG-rich domains can undergo liquid-liquid phase separation, creating a selective barrier.

  • Antibodies targeting specific epitopes within these domains are being used to study how post-translational modifications affect phase separation properties and transport selectivity.

• Single-molecule tracking:

  • Antibody fragments against NUP98 are being employed in single-molecule tracking experiments to monitor the dynamics of individual NUP98 molecules within the pore.

  • This has revealed unexpected mobility of NUP98 within seemingly stable structures, challenging static models of nuclear pore organization .

• Viral nuclear entry studies:

  • NUP98 antibodies are being used to investigate how viruses exploit the nuclear pore for nuclear entry.

  • Recent findings show that NUP98 interacts with HIV-1 capsid protein P24 and nucleocapsid protein P7, potentially promoting integration of the virus in the host nucleus .

• Correlative cryo-electron microscopy:

  • Immunogold labeling with NUP98 antibodies is being combined with cryo-electron tomography to map the precise location of NUP98 within the native architecture of the nuclear pore complex.

  • This approach is revealing previously unappreciated structural roles of NUP98 at the molecular level.

What emerging applications of NUP98 antibodies are being developed for cancer research and diagnostics?

Several innovative applications of NUP98 antibodies are emerging in cancer research and diagnostics:

• Multi-parameter flow cytometry panels:

  • NUP98 antibodies are being incorporated into flow cytometry panels for leukemia classification.

  • Altered NUP98 expression or subcellular distribution can help identify leukemia subtypes bearing NUP98 gene rearrangements, even when genetic testing is unavailable.

• Liquid biopsy development:

  • Research is exploring whether NUP98 fusion proteins can be detected in circulating tumor cells or plasma using highly sensitive antibody-based capture and detection systems.

  • This could enable non-invasive monitoring of disease progression and treatment response.

• Patient-derived xenograft (PDX) model characterization:

  • NUP98 antibodies are being used to validate PDX models of NUP98-rearranged leukemias.

  • By confirming the expression and localization of NUP98 fusion proteins in these models, researchers can ensure they accurately recapitulate human disease for therapeutic testing.

• Antibody-drug conjugates (ADCs):

  • Exploratory research is investigating whether NUP98 antibodies can be used to develop ADCs targeting cells with aberrant NUP98 expression or localization.

  • While challenging due to the predominantly intracellular localization of NUP98, certain disease states may present unique targetable epitopes.

• Combination with genomic profiling:

  • Integration of NUP98 immunostaining with next-generation sequencing is creating comprehensive diagnostic workflows.

  • This combined approach helps identify cryptic NUP98 rearrangements that may be missed by standard cytogenetic methods .

• Therapeutic response monitoring:

  • NUP98 antibodies are being used to track changes in NUP98 fusion protein expression during treatment.

  • Persistence of abnormal NUP98 staining patterns correlates with treatment resistance in some studies.

• Multiplex immunohistochemistry panels:

  • Development of multiplexed panels including NUP98 and its common fusion partners enables visualization of aberrant co-localization in tissue sections.

  • This approach provides spatial context for understanding the impact of NUP98 rearrangements on tissue architecture and cellular microenvironments.

• Single-cell protein analysis:

  • Combining NUP98 antibodies with single-cell proteomic methods like mass cytometry (CyTOF) allows researchers to characterize rare subpopulations of cells with altered NUP98 expression.

  • This is particularly valuable for identifying minor clones that may drive disease progression or relapse.

What new developments in antibody technology might enhance future NUP98 research?

Emerging antibody technologies promise to significantly advance NUP98 research in several ways:

• Single-domain antibodies and nanobodies:

  • These smaller antibody fragments derived from camelid heavy-chain antibodies can access epitopes in confined spaces.

  • For NUP98 research, nanobodies could provide better access to the dense environment of the nuclear pore complex, enabling more precise localization and functional studies with less steric hindrance .

• Genetically encoded intrabodies:

  • By expressing functional antibody fragments intracellularly, researchers can track and potentially modulate NUP98 in living cells.

  • This approach circumvents the need for cell permeabilization and fixation, preserving native dynamics and interactions.

• Proximity labeling antibodies:

  • Antibodies conjugated to enzymes like BioID or APEX2 can biotinylate proteins in close proximity to NUP98 when expressed in living cells.

  • This technology enables mapping of the local NUP98 interaction landscape with temporal precision, revealing dynamic changes in the NUP98 microenvironment.

• Bispecific antibodies:

  • Antibodies designed to simultaneously bind NUP98 and another protein of interest can be used to study specific interaction partners.

  • For leukemia research, bispecific antibodies targeting NUP98 and common fusion partners could provide unique reagents for detecting specific fusion proteins.

• Site-specific antibody conjugation:

  • Advanced conjugation chemistries enable precise attachment of labels at specific sites on antibodies.

  • For super-resolution microscopy of NUP98, this allows optimal placement of fluorophores to minimize the displacement between the label and the actual epitope position.

• Synthetic antibody libraries:

  • Rationally designed antibody libraries can be screened to generate antibodies against specific functional domains of NUP98.

  • This could yield reagents that selectively recognize different conformational states or post-translational modifications of NUP98.

• Recombinant antibody engineering:

  • Antibody affinity, specificity, and stability can be optimized through protein engineering.

  • For NUP98 research across species, engineered antibodies with broader cross-reactivity could facilitate evolutionary studies of nuclear pore complex structure and function .

• Antibody-based optogenetic tools:

  • Light-responsive antibody systems could allow temporal control over NUP98 interactions or functions.

  • This would enable precise perturbation experiments to dissect the kinetics of NUP98-dependent processes in nuclear transport and gene regulation.