U2AF1L4 Antibody

What is U2AF1L4 and what cellular functions does it perform?

U2AF1L4 (U2 small nuclear RNA auxiliary factor 1-like 4) is an RNA-binding protein that functions as a pre-mRNA splicing factor. It plays a critical role in both constitutive and enhancer-dependent splicing by mediating protein-protein interactions and protein-RNA interactions required for accurate 3'-splice site selection. The protein enhances the binding of U2AF2 to weak pyrimidine tracts and participates in the regulation of alternative pre-mRNA splicing. Notably, U2AF1L4 activates exon 5 skipping of PTPRC during T-cell activation, an event that can be reversed by GFI1. At the molecular level, it binds to RNA at the AG dinucleotide at the 3'-splice site, showing a preference for AGC or AGA sequences .

What types of U2AF1L4 antibodies are currently available for research?

Research-grade U2AF1L4 antibodies are available in several formats, including both polyclonal and monoclonal variants. Polyclonal antibodies are produced in various host animals, predominantly rabbits, and target different epitopes of the U2AF1L4 protein . Monoclonal antibodies, such as the EPR14349 clone, offer higher specificity to particular epitopes . Most available antibodies are unconjugated, though some can be obtained in conjugation-ready formats for fluorochromes, metal isotopes, oligonucleotides, and enzymes, making them suitable for various applications including antibody labeling, functional assays, cell-based assays, flow-based assays, and multiplex imaging applications . The immunogens used to generate these antibodies typically include recombinant human U2AF1L4 protein fragments, such as amino acids 1-202 .

What are the standard applications for U2AF1L4 antibodies in biological research?

U2AF1L4 antibodies are utilized across multiple experimental applications in biological research. The most common applications include Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), immunocytochemistry/immunofluorescence (ICC/IF), and flow cytometry (intracellular staining) . These applications allow researchers to detect, visualize, and quantify U2AF1L4 expression in various sample types. Western blotting enables protein expression level assessment, while IHC and ICC/IF provide spatial information about protein localization within tissues and cells, respectively. Flow cytometry allows for quantitative analysis of U2AF1L4 expression at the single-cell level. ELISA applications permit sensitive quantification of the protein in solution-based samples .

How should I optimize antibody dilutions for Western blotting with U2AF1L4 antibodies?

Optimizing antibody dilutions for Western blotting with U2AF1L4 antibodies requires a systematic approach. Begin with the manufacturer's recommended dilution range, typically 1:500-1:2000 for polyclonal antibodies . The optimization process should include:

• Initial titration experiment: Prepare a series of dilutions (e.g., 1:500, 1:1000, 1:2000) and test them on identical protein samples.

• Positive control inclusion: Include cell lines known to express U2AF1L4 (many human cell lines including HeLa are appropriate).

• Blocking optimization: Use 5% non-fat dry milk or BSA in TBST for reducing background.

• Incubation conditions: Test both 1-hour room temperature and overnight 4°C incubations to determine optimal signal-to-noise ratio.

• Signal development: Adjust exposure times based on signal intensity.

The optimal dilution will provide clear specific bands at the expected molecular weight (approximately 22-26 kDa) with minimal background . For monoclonal antibodies, start with higher dilutions (1:1000-1:5000) as they typically provide higher specificity with less background .

What controls should be included when using U2AF1L4 antibodies in immunofluorescence experiments?

When conducting immunofluorescence experiments with U2AF1L4 antibodies, proper controls are essential for accurate interpretation of results:

• Positive tissue/cell control: Include samples known to express U2AF1L4, such as HeLa cells or human tissue samples with documented expression .

• Negative controls:

  • Primary antibody omission: Apply only secondary antibody to detect non-specific binding.

  • Isotype control: Use non-specific IgG from the same host species at identical concentration.

  • Pre-adsorption control: Pre-incubate the antibody with excess U2AF1L4 recombinant protein to confirm specificity.

• Subcellular localization control: Co-stain with nuclear markers (e.g., DAPI) to confirm the expected nuclear/nuclear speckle localization of U2AF1L4 .

• Signal specificity control: Perform siRNA knockdown of U2AF1L4 to demonstrate reduced signal intensity.

For optimal results, fixation with 4% paraformaldehyde and permeabilization with 0.1-0.5% Triton X-100 is recommended. Expected staining pattern should show nuclear localization with enrichment in nuclear speckles, and potentially some cytoplasmic staining as U2AF1L4 displays active nucleo-cytoplasmic shuttling .

What is the recommended protocol for immunohistochemical detection of U2AF1L4 in tissue samples?

For immunohistochemical detection of U2AF1L4 in tissue samples, the following protocol is recommended:

• Sample preparation:

  • Fix tissue samples in 10% neutral buffered formalin.

  • Embed in paraffin and section at 4-6 μm thickness.

• Antigen retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) is typically effective.

  • Boil sections for 15-20 minutes followed by cooling at room temperature.

• Blocking and antibody application:

  • Block endogenous peroxidase activity with 3% H₂O₂.

  • Block non-specific binding with 5-10% normal serum from the species of the secondary antibody.

  • Apply primary U2AF1L4 antibody at dilutions between 1:20-1:200 .

  • Incubate overnight at 4°C or 1-2 hours at room temperature.

• Detection and visualization:

  • Apply appropriate secondary antibody and develop using DAB or other chromogens.

  • Counterstain with hematoxylin for nuclear visualization.

• Controls:

  • Include tissue sections known to express U2AF1L4.

  • Include negative controls (primary antibody omission and isotype controls).

The expected staining pattern is predominantly nuclear, with particular enrichment in nuclear speckles. Optimization may be required for different tissue types, and antigen retrieval conditions may need adjustment based on fixation duration and tissue type .

What species reactivity has been confirmed for U2AF1L4 antibodies, and how should cross-reactivity be validated?

Confirmed species reactivity for U2AF1L4 antibodies varies by product, with many antibodies showing validated reactivity to human, mouse, and rat samples . Some antibodies demonstrate broader cross-reactivity with species such as dog, cow, guinea pig, horse, rabbit, zebrafish, bat, chicken, hamster, monkey, and pig, though validation levels may vary .

To properly validate cross-reactivity:

• Sequence homology assessment: Compare the amino acid sequence of the immunogen with the orthologous sequence in the target species. High homology (>85%) suggests potential cross-reactivity.

• Empirical validation methods:

  • Western blot analysis with tissue/cell lysates from the species of interest, looking for bands of the expected molecular weight (approximately 22 kDa).

  • Immunohistochemistry/immunofluorescence on known positive tissues from the target species.

  • Inclusion of appropriate positive and negative controls from the target species.

• Antibody competition assays: Test if orthologous primate sequences can compete with human U2AF1L4 for binding with the antibody, as demonstrated in previous research .

When working with a new species, researchers should first conduct pilot experiments with positive controls to confirm reactivity before proceeding with experimental samples .

How do isoform differences in U2AF1L4 affect antibody selection and experimental design?

Isoform differences in U2AF1L4 have significant implications for antibody selection and experimental design. U2AF1L4 is known to have multiple isoforms with tissue-specific expression patterns. For example, isoform 2 is widely expressed across tissues, while isoform 3 shows high expression in heart, brain, and lung, lower expression in thymus, and much lower expression in peripheral blood leukocytes .

When selecting antibodies:

• Epitope targeting: Determine which region of U2AF1L4 the antibody targets. Antibodies raised against common regions will detect multiple isoforms, while those targeting unique regions will be isoform-specific.

• Application considerations:

  • For studies requiring detection of all isoforms, select antibodies targeting conserved regions.

  • For isoform-specific studies, verify that the antibody's epitope is unique to the isoform of interest.

• Experimental design adjustments:

  • Include positive controls expressing the specific isoform(s) of interest.

  • When analyzing tissues or cells, account for potential isoform expression differences.

  • Consider using primers or antibodies that can distinguish between isoforms for comprehensive analysis.

• Data interpretation:

  • Differences in apparent molecular weight on Western blots may reflect different isoforms rather than non-specific binding.

  • Variations in staining patterns across tissues may reflect isoform distribution differences.

This information is crucial for experiments involving tissues with known differential isoform expression, such as comparing neural tissues with peripheral tissues .

How can U2AF1L4 antibodies be used to investigate splicing mechanisms in disease models?

U2AF1L4 antibodies can be instrumental in investigating splicing mechanisms in disease models through several advanced approaches:

• Chromatin immunoprecipitation (ChIP) coupled with sequencing (ChIP-seq):

  • Use U2AF1L4 antibodies to identify genomic binding sites.

  • Compare binding patterns between normal and disease models to identify differential recruitment to splice sites.

• RNA immunoprecipitation (RIP) and crosslinking immunoprecipitation (CLIP):

  • Apply U2AF1L4 antibodies to isolate RNA-protein complexes.

  • Identify RNA targets and binding motifs that may be altered in disease states.

  • Compare binding preferences (e.g., AGC or AGA sequences) between normal and pathological conditions .

• Co-immunoprecipitation (Co-IP) assays:

  • Investigate protein-protein interactions involving U2AF1L4 in the spliceosome.

  • Examine how these interactions may be disrupted in disease states.

  • Study interactions with U2AF2 and how U2AF1L4 enhances its binding to weak pyrimidine tracts .

• Proximity ligation assays (PLA):

  • Visualize and quantify protein-protein interactions involving U2AF1L4 in situ.

  • Compare interaction frequencies and localizations between normal and disease samples.

• CRISPR-Cas9-mediated genome editing combined with immunofluorescence:

  • Create disease-relevant mutations in U2AF1L4.

  • Use antibodies to study changes in localization or expression patterns.

These approaches can reveal how alterations in U2AF1L4 function contribute to splicing dysregulation in diseases such as cancer, neurological disorders, and immune dysfunction, particularly focusing on its role in PTPRC exon 5 skipping during T-cell activation .

What role does U2AF1L4 play in alternative splicing regulation, and how can antibodies help elucidate these mechanisms?

U2AF1L4 plays a significant role in alternative splicing regulation through several mechanisms that can be investigated using antibodies:

• Regulation of exon inclusion/exclusion:

  • U2AF1L4 has been shown to activate exon 5 skipping of PTPRC during T-cell activation, a process that can be reversed by GFI1 .

  • Antibodies can be used in RNA-protein immunoprecipitation assays to identify the spectrum of exons regulated by U2AF1L4 binding.

• Splice site recognition:

  • U2AF1L4 binds to the AG dinucleotide at 3'-splice sites with preference for AGC or AGA sequences .

  • ChIP-seq and CLIP-seq experiments using U2AF1L4 antibodies can map binding sites genome-wide to identify regulated splice sites.

• Interactions with splicing enhancers:

  • As suggested by research on the enhancer element in the second intron of the U2AF1L4 gene itself , U2AF1L4 may interact with enhancer elements.

  • Antibodies can be used in DNA-protein interaction studies to investigate how these interactions affect alternative splicing decisions.

• Dynamic nuclear-cytoplasmic shuttling:

  • U2AF1L4 displays active nucleo-cytoplasmic shuttling, with interaction with C1qbp required for nuclear translocation .

  • Immunofluorescence using U2AF1L4 antibodies can track its localization under different cellular conditions or stimuli that affect splicing patterns.

• Methodological approaches:

  • Proximity-dependent biotin identification (BioID) coupled with U2AF1L4 antibodies can identify novel protein interactions in the splicing regulatory network.

  • Pulse-chase experiments with antibody detection can determine the dynamic assembly and disassembly of splicing complexes.

By employing these antibody-based techniques, researchers can elucidate how U2AF1L4 contributes to splicing decisions in normal cellular processes and how dysregulation may contribute to disease states .

How can researchers effectively use U2AF1L4 antibodies in multi-protein complex studies of the spliceosome?

Researchers can effectively use U2AF1L4 antibodies in multi-protein complex studies of the spliceosome through several specialized approaches:

• Sequential immunoprecipitation (IP) strategies:

  • Primary IP with U2AF1L4 antibody followed by secondary IP with antibodies against other spliceosome components.

  • This approach isolates specific subcomplexes containing U2AF1L4 and provides insights into assembly hierarchies.

• Proximity-based protein interaction mapping:

  • BioID or APEX2 proximity labeling fused to U2AF1L4, followed by antibody-based pulldown of biotinylated proteins.

  • Identification of proteins in close proximity to U2AF1L4 within the spliceosome in living cells.

• Mass spectrometry integration:

  • IP with U2AF1L4 antibodies followed by mass spectrometry analysis.

  • Create an interaction map of U2AF1L4 within different spliceosomal complexes under various conditions.

• Fluorescence microscopy techniques:

  • Immunofluorescence with U2AF1L4 antibodies combined with other spliceosome component antibodies.

  • Super-resolution microscopy or FRET analysis to study nanoscale organization and dynamics.

• In vitro reconstitution experiments:

  • Use purified components and U2AF1L4 antibodies to study assembly order and requirements.

  • Analyze how U2AF1L4 enhances the binding of U2AF2 to weak pyrimidine tracts .

• Experimental considerations:

  • Gentle lysis conditions to preserve native protein complexes (e.g., digitonin or NP-40 lysis).

  • Use of crosslinking agents (formaldehyde or DSP) to stabilize transient interactions.

  • RNase treatment controls to distinguish RNA-dependent from direct protein-protein interactions.

This multimodal approach allows researchers to decipher the role of U2AF1L4 within the complex architecture of the spliceosome and its contribution to splicing regulation .

What are common issues encountered with U2AF1L4 antibodies in Western blotting and how can they be resolved?

When working with U2AF1L4 antibodies in Western blotting, researchers may encounter several common issues that can be systematically addressed:

• Multiple bands or unexpected molecular weight:

  • Expected molecular weight for U2AF1L4 is approximately 22 kDa (202 amino acids) .

  • Resolution approach: Include positive controls with known U2AF1L4 expression, consider the possibility of detecting different isoforms, use gradient gels (10-20%) for better separation of lower molecular weight proteins.

• Weak or no signal:

  • Resolution approaches:

    • Increase antibody concentration (try 1:500 instead of 1:2000) .

    • Extend primary antibody incubation time (overnight at 4°C).

    • Enhance antigen retrieval by using fresh SDS-PAGE samples with minimal freeze-thaw cycles.

    • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity.

    • Increase protein loading (50-100 μg total protein per lane).

• High background:

  • Resolution approaches:

    • Increase blocking time or concentration (5% BSA or milk).

    • Add 0.1-0.3% Tween-20 to washing buffers.

    • Dilute antibody in fresh blocking buffer.

    • Extend washing steps (5x 5 minutes).

    • For polyclonal antibodies, pre-adsorb with non-specific proteins.

• Non-specific binding:

  • Resolution approaches:

    • Use monoclonal antibodies which typically offer higher specificity .

    • Validate specificity with siRNA knockdown controls.

    • Optimize antibody dilution through titration experiments.

• Storage-related issues:

  • U2AF1L4 antibodies should be stored at -20°C and are typically stable for one year after shipment .

  • Avoid repeated freeze-thaw cycles by preparing small aliquots.

  • For long-term storage, use storage buffer with 50% glycerol and 0.02% sodium azide at pH 7.3 .

These troubleshooting approaches should be implemented systematically, changing one variable at a time to identify the optimal conditions for your specific experimental system.

How should researchers analyze contradictory results when using different U2AF1L4 antibodies?

When facing contradictory results with different U2AF1L4 antibodies, researchers should employ a structured analysis approach:

• Epitope mapping comparison:

  • Compare the immunogen sequences of the antibodies (e.g., antibodies targeting AA 1-202 vs. AA 23-72) .

  • Different epitopes may reveal different aspects of protein function or accessibility in various experimental conditions.

• Antibody validation hierarchy:

  • Establish a validation hierarchy using orthogonal techniques:

    • Genetic controls: siRNA/shRNA knockdown or CRISPR knockout of U2AF1L4.

    • Overexpression controls: Exogenous expression of tagged U2AF1L4.

    • Peptide competition assays to confirm specificity.

• Technical parameter evaluation:

  • Compare antibody properties:

    • Polyclonal vs. monoclonal (clone EPR14349 vs. polyclonal antibodies) .

    • Host species (rabbit vs. mouse or goat).

    • Purification method (antigen affinity purification is preferred) .

• Application-specific optimization:

  • Some antibodies perform better in certain applications:

    • Compare manufacturer recommendations for each application (WB, IF, IHC, etc.).

    • Optimize each antibody individually for the specific application.

• Experimental interpretation:

  • Consider protein context dependencies:

    • Isoform-specific detection.

    • Post-translational modifications that may mask epitopes.

    • Protein-protein interactions affecting epitope accessibility.

    • Subcellular localization differences (nuclear vs. cytoplasmic fractions) .

• Consensus approach:

  • When possible, use multiple antibodies targeting different epitopes.

  • Compare results with literature data on U2AF1L4 expression and function.

  • Consider mass spectrometry validation for definitive protein identification.

By systematically analyzing these factors, researchers can resolve contradictions and gain more comprehensive insights into U2AF1L4 biology .

How should changes in U2AF1L4 expression be quantified and normalized in Western blot analysis?

Accurate quantification and normalization of U2AF1L4 expression in Western blot analysis requires a systematic approach:

• Image acquisition considerations:

  • Capture images within the linear dynamic range of the detection system.

  • Avoid saturated pixels that compromise quantification accuracy.

  • Use a digital imaging system rather than film for more precise quantification.

• Quantification methodology:

  • Measure integrated density (area × mean intensity) of U2AF1L4 bands using image analysis software (ImageJ, Image Studio, etc.).

  • Subtract local background from each lane individually.

  • For U2AF1L4, the expected molecular weight is approximately 22 kDa (202 amino acids) .

• Normalization strategies:

  • Primary normalization to housekeeping proteins:

    • GAPDH (37 kDa) or β-actin (42 kDa) for whole cell lysates.

    • Lamin A/C for nuclear fractions (particularly important since U2AF1L4 localizes primarily to the nucleus) .

    • α-Tubulin for cytoplasmic fractions.

  • Alternative normalization approaches:

    • Total protein normalization using stain-free gels or Ponceau S staining.

    • Loading control cocktails containing multiple housekeeping proteins.

    • Consider that traditional housekeeping genes may be affected by experimental conditions.

• Statistical analysis:

  • Perform at least three biological replicates for statistical validity.

  • Apply appropriate statistical tests (t-test, ANOVA) based on experimental design.

  • Report fold changes relative to control conditions with error bars.

  • Consider logarithmic transformation for fold changes spanning several orders of magnitude.

• Special considerations for U2AF1L4:

  • Account for potential isoform differences (isoform 2 is widely expressed while isoform 3 shows tissue-specific expression) .

  • Consider nucleo-cytoplasmic shuttling when interpreting changes in different cellular fractions .

  • Evaluate the possibility of post-translational modifications affecting antibody recognition.

This comprehensive approach ensures reliable quantification and biologically meaningful interpretation of changes in U2AF1L4 expression levels .

What insights can immunolocalization studies provide about U2AF1L4 function in different cellular contexts?

Immunolocalization studies using U2AF1L4 antibodies can provide critical insights into its function across various cellular contexts:

• Subcellular distribution patterns:

  • U2AF1L4 primarily localizes to the nucleus and nuclear speckles, consistent with its role in splicing .

  • It also displays active nucleo-cytoplasmic shuttling, suggesting potential extra-nuclear functions .

  • Changes in this distribution pattern can indicate altered functionality or stress responses.

• Co-localization analysis:

  • Co-localization with other splicing factors (e.g., U2AF2) can reveal functional complexes.

  • Quantitative co-localization metrics (Pearson's coefficient, Manders' overlap) provide objective measures of spatial relationships.

  • Differential co-localization across cell types may indicate tissue-specific splicing regulation.

• Dynamic changes during cellular processes:

  • Cell cycle-dependent localization changes may indicate cell cycle-specific splicing regulation.

  • Stress-induced relocalization (e.g., heat shock, oxidative stress) suggests roles in stress response pathways.

  • Monitoring localization during T-cell activation can provide insights into its role in PTPRC alternative splicing .

• Pathological alterations:

  • Aberrant localization in disease models may indicate dysregulation.

  • Correlation of mislocalization with splicing defects can establish causative relationships.

• Interaction-dependent localization:

  • The interaction with C1qbp is required for nuclear translocation of U2AF1L4 .

  • Disruption of this interaction may affect its localization and consequently its function.

• Methodological considerations:

  • Super-resolution microscopy (STED, STORM, SIM) provides nanoscale resolution of nuclear speckle organization.

  • Live-cell imaging with fluorescently tagged antibody fragments can capture dynamic changes.

  • Fluorescence recovery after photobleaching (FRAP) using labeled antibodies can assess protein mobility within compartments.

These immunolocalization studies can reveal functional states of U2AF1L4 that biochemical approaches might miss, particularly regarding its spatial and temporal regulation in different cellular contexts .

What emerging technologies could enhance U2AF1L4 antibody-based research in the future?

Several emerging technologies hold promise for advancing U2AF1L4 antibody-based research:

• Advanced spatial biology techniques:

  • Highly multiplexed imaging (CODEX, MIBI, Imaging Mass Cytometry) allowing simultaneous detection of U2AF1L4 with dozens of other proteins.

  • Spatial transcriptomics combined with antibody detection to correlate U2AF1L4 protein localization with splicing outcomes.

  • Super-resolution microscopy techniques (MINFLUX, STORM) achieving resolution below 10 nm to study nanoscale organization of splicing complexes.

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) using metal-conjugated U2AF1L4 antibodies for high-dimensional single-cell analysis.

  • Microfluidic antibody-based proteomics to quantify U2AF1L4 and interaction partners in individual cells.

  • Integration with single-cell transcriptomics to correlate U2AF1L4 protein levels with splicing patterns.

• Engineered antibody technologies:

  • Nanobodies or single-domain antibodies against U2AF1L4 for improved penetration in tissues and live-cell imaging.

  • Split-antibody complementation systems to visualize U2AF1L4 interactions in living cells.

  • Conditionally stable antibody fragments that only function in specific cellular compartments.

• Proximity labeling advancements:

  • TurboID or miniTurbo fusions with antibody-based targeting to map the U2AF1L4 interactome with temporal control.

  • APEX2-antibody conjugates for electron microscopy visualization of U2AF1L4 ultrastructural localization.

• Functional antibody applications:

  • Optogenetic control of antibody binding to manipulate U2AF1L4 function with spatiotemporal precision.

  • Degron-tagged antibody fragments to induce targeted degradation of U2AF1L4.

  • CRISPR-based protein tagging for endogenous labeling compatible with antibody detection.

• Computational integration:

  • Machine learning algorithms for automated analysis of U2AF1L4 localization patterns across large datasets.

  • Integrative multi-omics approaches combining antibody-based detection with transcriptomics and genomics.

These technologies have the potential to transform our understanding of U2AF1L4's role in splicing regulation and disease pathogenesis by providing unprecedented resolution, specificity, and functional insights .

How might U2AF1L4 antibodies contribute to understanding the role of this protein in disease pathogenesis?

U2AF1L4 antibodies can significantly advance our understanding of this protein's role in disease pathogenesis through multiple research avenues:

• Cancer research applications:

  • Immunohistochemical profiling of U2AF1L4 expression across tumor types and correlation with clinical outcomes.

  • Investigation of U2AF1L4-mediated alternative splicing events that might promote oncogenesis.

  • Analysis of U2AF1L4 subcellular localization changes during malignant transformation.

  • Comparison with related splicing factors like U2AF1, which has known mutations in myelodysplastic syndromes and leukemias.

• Immunological disorder investigations:

  • Given U2AF1L4's role in PTPRC (CD45) alternative splicing during T-cell activation , antibodies can help investigate:

    • T-cell dysfunction in autoimmune diseases.

    • Altered splicing regulation in primary immunodeficiencies.

    • Changes in U2AF1L4 function during immune responses to pathogens.

• Neurodegenerative disease research:

  • Examination of U2AF1L4 expression and localization in brain tissues from patients with neurodegenerative disorders.

  • Investigation of altered splicing patterns in disease-relevant genes.

  • Correlation of U2AF1L4 dysfunction with RNA processing defects seen in many neurological conditions.

• High-throughput screening approaches:

  • Development of antibody-based assays for screening compounds that modulate U2AF1L4 function or localization.

  • Identification of molecules that could correct aberrant splicing patterns in disease models.

• Mechanistic insights through protein interaction studies:

  • Investigation of how disease-associated mutations in other splicing factors affect interaction with U2AF1L4.

  • Examination of how stress conditions alter the U2AF1L4 interactome in disease contexts.

  • Study of how post-translational modifications of U2AF1L4 might be dysregulated in pathological states.

• Therapeutic development:

  • Use of antibodies to validate U2AF1L4 as a potential therapeutic target in diseases involving splicing dysregulation.

  • Development of antibody-drug conjugates for targeted delivery to cells with aberrant U2AF1L4 expression.

  • Creation of intrabodies to modulate U2AF1L4 function in disease models.

These approaches can collectively illuminate the complex roles of U2AF1L4 in disease pathogenesis and potentially identify novel therapeutic strategies .

What are the key considerations for selecting the optimal U2AF1L4 antibody for specific research questions?

Selecting the optimal U2AF1L4 antibody requires careful consideration of multiple factors tailored to specific research questions:

• Application-specific selection:

  • For protein quantification (Western blot/ELISA): Select antibodies validated specifically for these applications with demonstrated linearity of detection .

  • For localization studies (IHC/IF): Choose antibodies with low background and specific nuclear/nuclear speckle staining pattern .

  • For protein interactions (IP/Co-IP): Select antibodies that don't interfere with protein-protein interaction domains.

  • For flow cytometry: Use antibodies specifically validated for intracellular staining protocols .

• Technical specifications:

  • Monoclonal vs. polyclonal: Monoclonal antibodies (like EPR14349) offer higher specificity but may be sensitive to epitope modifications; polyclonal antibodies provide robust detection but potentially higher background .

  • Host species: Consider compatibility with other antibodies in multi-labeling experiments.

  • Isotype: Typically IgG for most applications, but may affect secondary antibody selection .

  • Epitope location: Antibodies targeting different regions (e.g., AA 1-202 vs. 23-72) may reveal different aspects of protein biology .

• Experimental system considerations:

  • Species reactivity: Ensure antibody reactivity matches your experimental system (human, mouse, rat, etc.) .

  • Isoform detection: For comprehensive studies, select antibodies recognizing all isoforms; for isoform-specific research, choose antibodies targeting unique regions .

  • Post-translational modifications: Consider whether modifications might mask epitopes in your experimental context.

• Validation rigor:

  • Prioritize antibodies validated by multiple methods (WB, IP, IF, KO controls).

  • Review validation data for relevant cell/tissue types.

  • Consider independent validation using genetic approaches (siRNA, CRISPR).

• Practical considerations:

  • Storage requirements and stability (typically -20°C storage with 50% glycerol) .

  • Cost-effectiveness for long-term projects.

  • Technical support availability from manufacturers.

By systematically evaluating these factors, researchers can select the U2AF1L4 antibody best suited to answer their specific research questions with maximum reliability and relevant biological insights .

How can researchers integrate U2AF1L4 antibody techniques with genomic and transcriptomic approaches for comprehensive splicing research?

Integrating U2AF1L4 antibody techniques with genomic and transcriptomic approaches enables comprehensive splicing research through multi-layered analysis:

• Integrative experimental design strategies:

  • Parallel sample processing for proteomics and transcriptomics from the same biological specimens.

  • Sequential analysis where antibody-based findings guide targeted genomic/transcriptomic investigations.

  • Multi-modal single-cell approaches combining protein detection with RNA analysis.

• Chromatin-focused integration:

  • ChIP-seq using U2AF1L4 antibodies to identify genomic binding sites.

  • Integration with RNA-seq to correlate binding with splicing outcomes.

  • Combination with ATAC-seq or DNase-seq to assess chromatin accessibility at U2AF1L4 binding sites.

  • Analysis of enhancer elements, similar to the one identified in the U2AF1L4 gene itself .

• RNA-protein interaction mapping:

  • CLIP-seq or eCLIP using U2AF1L4 antibodies to identify direct RNA targets.

  • Integration with RNA-seq to determine how binding correlates with splicing decisions.

  • Motif analysis to refine understanding of U2AF1L4's preference for AGC or AGA sequences .

• Protein interaction networks:

  • Immunoprecipitation with U2AF1L4 antibodies followed by mass spectrometry.

  • Integration with transcriptome-wide splicing analysis to correlate protein interactions with functional outcomes.

  • Proximity labeling approaches to identify context-specific interaction partners.

• Functional validation pipelines:

  • U2AF1L4 perturbation (knockdown/overexpression) followed by RNA-seq.

  • Antibody-based detection to confirm perturbation efficiency.

  • Integration with splicing-sensitive microarrays or targeted RT-PCR panels.

• Data integration frameworks:

  • Computational workflows linking antibody-derived protein localization data with RNA-seq splicing analysis.

  • Machine learning approaches to identify patterns connecting U2AF1L4 binding, protein interactions, and splicing outcomes.

  • Network analysis incorporating protein-protein, protein-RNA, and genetic interactions.

• Temporal dynamics assessment:

  • Time-course experiments capturing both protein-level changes (antibody detection) and RNA processing dynamics.

  • Pulse-chase approaches to track newly synthesized U2AF1L4 and correlate with emerging splicing patterns.