NXF5 Antibody

What is NXF5 and what cellular functions does it regulate?

NXF5 (Nuclear RNA Export Factor 5) is a member of the nuclear RNA export factor family that plays a crucial role in mRNA export from the nucleus to the cytoplasm, a fundamental process in gene expression regulation. Despite sharing homology with other family members, NXF5 has lost several C-terminal protein domains found in other family members that are required for export activity, and may be an evolving pseudogene . The protein contains common domain features including a noncanonical RNP-type RNA-binding domain (RBD), four leucine-rich repeats (LRRs), a nuclear transport factor 2 (NTF2)-like domain, and a ubiquitin-associated domain that mediates interactions with nucleoporins . Dysregulation of NXF5 has been linked to various diseases, including cancer and neurological disorders .

What types of NXF5 antibodies are currently available for research applications?

Several types of NXF5 antibodies are available for research applications:

• Polyclonal antibodies: Most commonly available, including:

  • PACO28686 from Assay Genie (rabbit host, reactive to human samples)

  • Abbexa's antibody (rabbit host, reactive to human samples)

  • ABIN2581725 (rabbit host, targeting amino acids 53-80)

• By host species:

  • Rabbit polyclonal antibodies are predominant

• By application specificity:

  • Western Blot (WB) validated antibodies

  • Immunohistochemistry (IHC) validated antibodies

  • Immunofluorescence (IF) validated antibodies

  • ELISA validated antibodies

Most commercially available NXF5 antibodies are unconjugated, though specific research needs may require customized conjugation approaches .

How does the molecular structure of NXF5 differ from other nuclear export factors?

NXF5 differs from other nuclear export factors primarily through the loss of several C-terminal protein domains that are typically required for full export activity in other family members . While it maintains the noncanonical RNP-type RNA-binding domain (RBD) and the four leucine-rich repeats (LRRs) common to this family, its functional capacity may be altered due to these structural differences .

The protein contains an NTF2-like domain that allows for heterodimerization with NTF2-related export protein-1 (NXT1), and a ubiquitin-associated domain mediating interactions with nucleoporins . These structural features are important for researchers to consider when designing experiments targeting specific domains of the protein. The calculated molecular weight of human NXF5 is approximately 45.6 kDa , which should be considered when analyzing western blot results.

What are the optimal conditions for using NXF5 antibodies in western blotting experiments?

For western blotting applications with NXF5 antibodies, researchers should consider the following protocol parameters:

When running western blots, it's crucial to include appropriate positive controls. For NXF5 detection, HL60 whole cell lysate has been validated as an effective positive control . The observation of a predominant 46 kDa band aligns with the calculated molecular weight of 45.6 kDa for the primary isoform .

How should researchers optimize immunofluorescence protocols when studying NXF5 localization?

For optimal immunofluorescence results when studying NXF5 cellular localization:

• Dilution optimization: Start with a dilution range of 1:50-1:200 for NXF5 antibodies such as PACO28686 . Perform a dilution series to determine optimal signal-to-noise ratio for your specific cell type.

• Cell fixation: Standard 4% paraformaldehyde fixation (10-15 minutes at room temperature) followed by permeabilization with 0.1-0.2% Triton X-100 works well for most nuclear proteins including NXF5.

• Validated cell lines: HepG2 cells have been successfully used for NXF5 immunofluorescence studies at 1:100 dilution . Consider using these as positive controls when establishing the protocol for your cell line of interest.

• Fluorophore selection: Secondary antibodies conjugated with Alexa Fluor 488 have been validated for NXF5 detection . This wavelength provides good signal separation from common nuclear counterstains like DAPI.

• Controls: Include a negative control (secondary antibody only) and if possible, a peptide competition assay using NXF5 blocking peptide to confirm specificity .

• Counterstaining: Since NXF5 is involved in nuclear export, nuclear counterstaining with DAPI is essential to confirm localization patterns at the nuclear envelope or within nuclear subcompartments.

• Confocal microscopy: Due to the specific subcellular localization patterns of NXF5, confocal microscopy is recommended over standard wide-field fluorescence for more precise localization data.

What sample preparation techniques yield optimal results for NXF5 detection in human tissues?

For optimal NXF5 detection in human tissue samples:

• Fixation: For formalin-fixed paraffin-embedded (FFPE) samples, standard 10% neutral buffered formalin fixation for 24-48 hours is recommended. Over-fixation can mask epitopes, particularly for nuclear proteins like NXF5.

• Antigen retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is typically effective for NXF5 detection. For challenging samples, consider EDTA buffer (pH 9.0) as an alternative.

• Blocking: For IHC applications using NXF5 antibodies (e.g., PACO28686), a 1:20-1:200 dilution range is recommended . Start with a 5% normal serum blocking step (from the same species as the secondary antibody) to reduce background.

• Fresh tissue handling: If working with fresh tissue samples rather than FFPE, rapid fixation or flash freezing is critical to preserve RNA-binding proteins like NXF5.

• Positive controls: Include tissue sections known to express NXF5, particularly those from tissues with high transcriptional activity.

• Peptide competition: Consider including a peptide competition control using a specific NXF5 blocking peptide, such as the C-terminal blocking peptide available for certain antibodies .

• Multiplexing considerations: When performing multiplexed immunohistochemistry to co-localize NXF5 with interaction partners, sequential staining protocols are recommended rather than cocktail approaches to minimize cross-reactivity.

What are common causes of non-specific binding when using NXF5 antibodies and how can they be addressed?

Common causes of non-specific binding with NXF5 antibodies include:

• Insufficient blocking: Increase blocking time (1-2 hours) or concentration (from 3% to 5% BSA/normal serum). For particularly problematic samples, consider dual blocking with both BSA and normal serum.

• Antibody cross-reactivity: NXF5 belongs to a family of related nuclear export factors. Verify antibody specificity through:

  • Peptide competition assays using specific NXF5 blocking peptides

  • Western blot analysis confirming the correct molecular weight (approximately 45.6 kDa)

  • Genetic validation in cell lines with NXF5 knockdown (using available NXF5 esiRNA products)

• High antibody concentration: Dilute antibodies according to manufacturer recommendations (e.g., 1:500-1:2000 for western blot, 1:20-1:200 for IHC with PACO28686) .

• Insufficient washing: Implement more stringent washing steps (e.g., increase number of washes or duration, or add low concentrations of detergent to wash buffers).

• Sample-specific issues: For challenging tissue types, optimize fixation and antigen retrieval methods. Consider testing both citrate and EDTA-based antigen retrieval protocols.

• Secondary antibody problems: Run secondary-only controls to identify background from this source. Consider using highly cross-adsorbed secondary antibodies.

• Endogenous peroxidase or phosphatase activity: For IHC applications, include appropriate quenching steps (e.g., 3% hydrogen peroxide for peroxidase-based detection systems).

How can researchers validate the specificity of their NXF5 antibody results?

Validating specificity of NXF5 antibody results requires a multi-faceted approach:

• Multiple antibody validation: Use antibodies targeting different epitopes of NXF5. Compare results between the N-terminal targeting antibody like ABIN2581725 (targeting AA 53-80) with antibodies targeting other regions.

• Peptide competition assays: Pre-incubate the antibody with a specific NXF5 blocking peptide, such as the C-terminal blocking peptide available for certain antibodies . Disappearance of signal confirms specificity.

• Genetic validation approaches:

  • siRNA/shRNA knockdown of NXF5 using commercially available reagents

  • CRISPR/Cas9-mediated knockout of NXF5

  • Overexpression of tagged NXF5 with co-localization studies

• Multiple detection methods: Confirm findings using orthogonal techniques (e.g., if using IHC, confirm with immunofluorescence or western blotting).

• Predicted molecular weight verification: Confirm observation of the expected 45.6 kDa band in western blots , with awareness of potential splice variants.

• Cross-species validation: If appropriate for your research question, examine conservation of staining patterns across species, noting that NXF5 antibodies have been validated in human samples primarily .

• Subcellular localization confirmation: Verify that observed localization patterns align with known biology of NXF5 (nuclear/nuclear envelope localization consistent with its role in mRNA export) .

How can NXF5 antibodies be effectively used to study its role in RNA export pathways in disease models?

For studying NXF5's role in RNA export pathways in disease models:

• Co-immunoprecipitation (Co-IP) approach:

  • Use NXF5 antibodies (e.g., PACO28686) for pull-down experiments to identify interaction partners

  • Compare binding partners between normal and disease models

  • Confirm interactions with known components of the nuclear export machinery (e.g., NXT1, nucleoporins)

  • Recommended antibody dilution: Start with manufacturer's recommendations for IP applications

• RNA immunoprecipitation (RIP) methodology:

  • Employ NXF5 antibodies to isolate RNA-protein complexes

  • Analyze bound RNAs through sequencing to identify transcripts preferentially exported by NXF5

  • Compare RNA binding profiles between normal and disease states

  • Critical control: Include IgG control and validation through qPCR of known targets

• Proximity ligation assay (PLA) application:

  • Use NXF5 antibodies in combination with antibodies against potential interaction partners

  • Quantify interactions at the single-cell level in tissue sections from disease models

  • Compare interaction frequencies between normal and diseased tissues

• Fluorescence recovery after photobleaching (FRAP):

  • Combine NXF5 antibody staining with live-cell imaging techniques

  • Measure dynamics of NXF5-containing complexes at the nuclear envelope

  • Compare kinetics between normal and disease model cells

• High-content screening approach:

  • Use NXF5 antibodies for immunofluorescence in large-scale cellular models

  • Quantify changes in localization, expression level, or interaction patterns following drug treatments

  • Identify compounds that normalize aberrant NXF5 function in disease models

• Spatial transcriptomics integration:

  • Combine NXF5 immunohistochemistry with spatial transcriptomics

  • Correlate NXF5 protein levels with localized mRNA export efficiency

  • Map alterations in RNA export patterns in disease tissues

What approaches can be used to study NXF5 interactions with other components of the mRNA export machinery?

To study interactions between NXF5 and other components of the mRNA export machinery:

• Structural domain mapping:

  • Use domain-specific antibodies or tagged truncation constructs

  • Focus on the known functional domains: RNP-type RNA-binding domain (RBD), leucine-rich repeats (LRRs), nuclear transport factor 2 (NTF2)-like domain, and ubiquitin-associated domain

  • Compare interaction patterns with those of other NXF family members that retain full export activity

• FRET/FLIM analysis:

  • Use antibodies against NXF5 and potential interaction partners labeled with appropriate fluorophore pairs

  • Measure energy transfer in fixed or live cells to detect proximity (<10nm) between proteins

  • Analyze changes in FRET efficiency under different cellular conditions (stress, differentiation, etc.)

• Bimolecular Fluorescence Complementation (BiFC):

  • Express NXF5 and potential partners as fusion proteins with complementary fragments of a fluorescent protein

  • Visualize interactions through reconstitution of fluorescence when proteins interact

  • Map interaction domains through mutation analysis

• Mass spectrometry after immunoprecipitation:

  • Use validated NXF5 antibodies for pull-down experiments

  • Identify binding partners through LC-MS/MS analysis

  • Compare interactome between different cell types or conditions

  • Quantify changes in interaction stoichiometry in disease models

• Crosslinking immunoprecipitation (CLIP) methods:

  • Utilize NXF5 antibodies to isolate crosslinked RNA-protein complexes

  • Identify both protein partners and bound RNA species

  • Map binding sites on RNA molecules through sequencing analysis

• Super-resolution microscopy:

  • Use high-affinity NXF5 antibodies for immunofluorescence

  • Employ STORM or STED microscopy to visualize nanoscale co-localization with nuclear pore components

  • Quantify spatial relationships at resolution beyond diffraction limit

• Computational modeling integration:

  • Use experimental data from antibody-based studies to inform protein-protein interaction models

  • Predict functional consequences of mutations or post-translational modifications

  • Design targeted interventions to modulate specific interactions

How should researchers interpret discrepancies between different antibody-based detection methods for NXF5?

When faced with discrepancies between different antibody-based detection methods for NXF5:

• Epitope accessibility considerations:

  • Different antibodies target distinct epitopes (e.g., ABIN2581725 targets AA 53-80 in the N-terminal region)

  • Fixation methods affect epitope accessibility differently across techniques

  • Potential solution: Use multiple antibodies targeting different regions of NXF5, or consider native vs. denatured conditions

• Splice variant detection:

  • NXF5 has multiple transcript variants, though most are candidates for nonsense-mediated decay

  • Western blot may detect multiple bands (observed bands at 46, 43, 20, 36, 35 kDa)

  • Potential solution: Use RNA-seq or RT-PCR to confirm expression of specific variants in your experimental system

• Technical variability sources:

  • Batch-to-batch variation in antibody production

  • Differences in detection sensitivity between methods

  • Potential solution: Validate new antibody lots against previously characterized samples

• Cross-reactivity with related proteins:

  • NXF5 shares homology with other nuclear export factors

  • Potential solution: Include positive controls with known expression patterns and negative controls where possible

• Quantification challenges:

  • Different methods have distinct dynamic ranges and quantitative capabilities

  • Potential solution: Use calibrated standards or spike-in controls for quantitative comparisons

• Proper statistical analysis:

  • Apply appropriate statistical tests for each data type

  • Consider non-parametric methods for immunohistochemistry scoring

  • Implement replicate experiments with power analysis for sample size determination

• Integration of multiple data sources:

  • Combine protein-level data (antibody-based) with transcript-level analysis (RNA-seq, qPCR)

  • Correlate findings with functional assays of mRNA export

What emerging research areas are investigating the therapeutic potential of targeting NXF5 and its pathways?

Emerging research areas investigating the therapeutic potential of targeting NXF5 and its pathways include:

• Neurological disorder interventions:

  • NXF5 dysregulation has been linked to neurological disorders

  • Research focus: Using NXF5 antibodies to characterize expression patterns in patient-derived tissues

  • Therapeutic approach: Modulating NXF5 function to restore proper mRNA export in affected neurons

• Cancer biology applications:

  • Altered RNA export machinery contributes to cancer progression

  • Research direction: Profiling NXF5 expression and localization across cancer types

  • Antibody application: Using NXF5 antibodies for tissue microarray analysis and patient stratification

• RNA therapeutics development:

  • Small molecule screening to identify compounds that modulate NXF5-mediated export

  • Research approach: Using NXF5 antibodies in high-content screening assays

  • Therapeutic goal: Selective modulation of export for specific transcripts

• Selective inhibitor development:

  • Structure-based design of inhibitors targeting NXF5-specific domains

  • Research need: High-resolution structural data on NXF5 domains and interactions

  • Verification approach: Using antibodies to confirm target engagement in cellular models

• Biomarker development:

  • Evaluation of NXF5 as a potential diagnostic or prognostic marker

  • Research methodology: Quantitative analysis of NXF5 expression in patient cohorts

  • Antibody application: Development of clinical-grade antibodies for diagnostic use

• Viral infection interventions:

  • Many viruses hijack nuclear export machinery for viral replication

  • Research direction: Characterizing NXF5 interactions with viral components

  • Therapeutic concept: Targeting virus-specific interactions with the export machinery

• Gene therapy considerations:

  • NXF5 as a potential target for enhancing therapeutic gene expression

  • Research approach: Modifying export efficiency of therapeutic transcripts

  • Verification method: Using antibodies to track subcellular localization of therapeutic mRNAs