ESF1.1 Antibody

What is ESF1 and what cellular functions does it regulate?

ESF1 (ESF1, Nucleolar Pre-rRNA Processing Protein, Homolog) is a protein critically involved in ribosome biogenesis, particularly in the early stages of this process which are less studied than later stages . Research indicates that ESF1 directly contributes to ribosomal subunit maturation by interacting with precursors of the small ribosomal subunits (40S) . Knockdown experiments demonstrate that ESF1 significantly alters the pattern of RNA products derived from 47S pre-rRNA, suggesting its essential role in pre-rRNA processing . In yeast models, ESF1 depletion leads to dramatic decreases in 27SA2 and 20S pre-rRNAs, with concurrent accumulation of 35S and aberrant 23S pre-ribosomal RNAs, indicating its potential involvement in A0, A1, and A2 site cleavage events . Beyond ribosome biogenesis, recent studies have identified ESF1 as a potential oncogenic factor, particularly in estrogen receptor-positive (ER+) breast cancer .

What are the key considerations when selecting an ESF1 antibody for research?

When selecting an ESF1 antibody, researchers should consider multiple factors to ensure experimental success:

• Target epitope: Determine whether you need an antibody targeting the C-terminal region or specific amino acid sequences (e.g., AA 1-851, AA 22-234, AA 801-851) based on your research question .

• Host species: Available options include rabbit and mouse-derived antibodies, which must be compatible with your experimental design and other antibodies used in multi-labeling procedures .

• Clonality: Choose between polyclonal antibodies (which recognize multiple epitopes) or monoclonal antibodies based on specificity requirements .

• Conjugation: Select appropriate conjugation (unconjugated, FITC, HRP, Biotin) based on your detection method .

• Validated applications: Ensure the antibody has been validated for your intended application (Western blotting, immunofluorescence, immunoprecipitation, ELISA) .

• Species cross-reactivity: Verify reactivity with your study species. For example, some ESF1 antibodies show different reactivity percentages (Dog: 79%, Horse: 79%, Human: 100%, Pig: 86%, Rabbit: 86%, Rat: 86%) .

• Validation data: Review available validation data such as Western blot images or immunofluorescence results provided by manufacturers .

How can I optimize ESF1 antibody dilution for immunofluorescence experiments?

Optimal dilution of ESF1 antibody for immunofluorescence requires methodical approach:

• Starting dilution reference: Published studies indicate 1:50 dilution has been successfully used for ESF1 antibody in immunofluorescent analysis of MCF-7 cells with Rhodamine-Goat anti-Rabbit IgG as secondary antibody .

• Titration protocol: Perform a dilution series (e.g., 1:25, 1:50, 1:100, 1:200) on identical samples to identify optimal signal-to-noise ratio.

• Cell fixation method: For ESF1 detection, acetone fixation at -20°C for 10 minutes has proven effective .

• Antibody incubation: Incubate cells with primary antibody (rabbit anti-ESF1) at room temperature for 1 hour, followed by three 5-minute PBS washes .

• Secondary antibody selection: Use fluorophore-conjugated secondary antibodies such as Alexa Fluor 568 goat anti-rabbit IgG (H+L) for detection .

• Co-localization studies: For nuclear/nucleolar localization confirmation, co-stain with established markers such as B23/nucleophosmin or SURF6 .

• Imaging parameters: Adjust exposure times and gain settings to prevent saturation while maintaining sufficient signal intensity.

• Positive controls: Include cells known to express ESF1 at high levels (e.g., MCF-7 breast cancer cells) .

How can ESF1 antibodies be utilized to investigate ribosome biogenesis pathways?

ESF1 antibodies serve as powerful tools for dissecting ribosome biogenesis mechanisms through several sophisticated approaches:

• Immunoprecipitation for RNA interaction studies: ESF1 antibodies can be employed in RNA immunoprecipitation (RIP) experiments to identify specific pre-rRNA species that associate with ESF1, similar to studies showing RPF1 interactions with 35S, 27SA, and 27SB pre-ribosomal RNAs .

• Polysome profiling analysis: Following cycloheximide treatment (100 μg/mL for 30 minutes), ESF1 antibodies can be used to track ESF1 association with specific ribosomal fractions separated by sucrose gradient ultracentrifugation to determine its presence in pre-40S particles .

• Co-immunoprecipitation for protein complex identification: ESF1 antibodies can pull down ESF1-containing complexes to identify protein partners involved in pre-rRNA processing, providing insight into the composition of pre-ribosomal particles.

• ChIP-seq applications: For investigating potential roles of ESF1 in regulating ribosomal DNA transcription.

• Proximity ligation assays: To visualize and quantify in situ interactions between ESF1 and other nucleolar proteins.

• CRISPR-Cas9 knockout validation: ESF1 antibodies are essential for confirming knockout efficiency and phenotypic consequences in gene editing experiments.

• Nucleolar stress response studies: Monitor ESF1 localization changes during various cellular stresses that impact ribosome biogenesis.

This multi-faceted approach allows comprehensive characterization of ESF1's role in the complex ribosome biogenesis pathway.

What are the critical considerations when using ESF1 antibodies for cancer research?

When employing ESF1 antibodies in cancer research, particularly for ER+ breast cancer studies, researchers should consider several sophisticated methodological aspects:

• Expression correlation with clinical outcomes: Recent proteomic analyses have identified ESF1 as a differentially expressed protein (DEP) in ER+ breast cancer compared to adjacent normal tissue, with upregulation associated with poorer patient prognosis .

• Functional validation protocols:

  • Knockdown verification: Western blot with ESF1 antibodies is essential to confirm siRNA/shRNA efficiency before phenotypic assays

  • Colony formation assays: Following ESF1 knockdown, these reveal proliferation impacts

  • Wound healing assays: ESF1 knockdown has been shown to inhibit this process, suggesting roles in migration

  • Apoptosis measurement: Flow cytometry following knockdown shows increased cell death

• Protein interaction network analysis: STRING analysis positions ESF1 as a hub gene in breast cancer, necessitating co-immunoprecipitation studies with ESF1 antibodies to identify cancer-specific interactions .

• Tissue microarray validation: When analyzing patient samples, optimize antibody dilution and staining protocols for tissue microarrays to ensure consistent immunohistochemical detection across specimens.

• Subcellular localization changes: Cancer cells may show altered ESF1 localization patterns compared to normal cells, requiring careful immunofluorescence optimization.

• Quantification methods: Establish consistent quantification parameters for Western blot and immunohistochemistry to enable statistical comparison across patient cohorts.

• Controls for specificity: Include appropriate controls to rule out non-specific binding, particularly in complex tumor microenvironments.

How can ESF1 antibody be used to investigate protein-protein interactions in nucleolar complexes?

Investigating ESF1's protein-protein interactions within nucleolar complexes requires sophisticated immunological approaches:

• Co-immunoprecipitation protocol optimization:

  • Cell lysis buffer selection: Use gentle non-ionic detergents (0.5% NP-40) to preserve nucleolar protein complexes

  • DNase/RNase treatment: Determine whether interactions are DNA/RNA-dependent by treating lysates with nucleases before immunoprecipitation

  • Cross-linking protocols: Employ formaldehyde (1%) or DSS cross-linking to capture transient interactions

  • Elution conditions: Optimize between native (competition with immunizing peptide) or denaturing conditions

• Proximity-dependent labeling techniques:

  • BioID approach: Generate ESF1-BioID fusion constructs to biotinylate proximal proteins

  • APEX2 system: ESF1-APEX2 fusions allow rapid biotinylation of neighboring proteins upon H₂O₂ exposure

  • Mass spectrometry analysis: Identify biotinylated proteins through LC-MS/MS

• Fluorescence resonance energy transfer (FRET):

  • Create ESF1-fluorophore fusion proteins (e.g., ESF1-CFP)

  • Generate potential interacting partner fusions with compatible fluorophores (e.g., SURF6-YFP)

  • Measure energy transfer to detect interactions within 10nm distance

• Split-complementation assays:

  • Luciferase complementation: Fuse ESF1 and candidate partners to luciferase fragments

  • BiFC technique: ESF1 fusions with split fluorescent protein segments

• Two-hybrid screening adaptations:

  • Nucleolar-specific yeast three-hybrid screening incorporating RNA components

  • Mammalian two-hybrid systems optimized for nucleolar proteins

These methods provide complementary approaches to map ESF1's interaction network in nucleolar complexes, with validation by reciprocal co-immunoprecipitation using antibodies against identified partners.

How can I resolve non-specific binding issues when using ESF1 antibody in Western blotting?

Non-specific binding in Western blotting with ESF1 antibodies can be systematically addressed through these methodological refinements:

• Blocking optimization:

  • Test different blocking agents: Compare 5% non-fat dry milk, 5% BSA, commercial blocking buffers

  • Blocking duration: Extend from standard 1 hour to overnight at 4°C

  • Add 0.1-0.3% Tween-20 to blocking solution to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Create a dilution series (1:500, 1:1000, 1:2000, 1:5000)

  • Validated dilution reference: 1:2000 has been successfully used for ESF1 antibody in mouse heart tissue Western blot

  • Dilute antibody in blocking buffer containing 0.05-0.1% Tween-20

• Washing protocol enhancement:

  • Increase washing frequency (5× 5 minutes instead of 3× 5 minutes)

  • Use TBS-T with 0.1% Tween-20 for stringent washing

  • Add 0.05% SDS to washing buffer for particularly stubborn non-specific binding

• Sample preparation refinement:

  • Include protease inhibitor cocktails during extraction

  • Perform additional centrifugation steps to remove insoluble material

  • Consider nuclear extraction protocols for cleaner ESF1 detection

• Controls implementation:

  • Use ESF1 knockdown/knockout samples as negative controls

  • Pre-absorb antibody with immunizing peptide if available

  • Include molecular weight marker to confirm specific band (ESF1: 851 AA)

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Reduce secondary antibody concentration

  • Consider secondary antibodies specifically optimized for Western blotting

• Membrane handling:

  • Cut membrane to include only relevant molecular weight ranges

  • Consider alternative membrane types (PVDF vs. nitrocellulose)

• Detection system optimization:

  • Compare ECL systems of different sensitivities

  • For fluorescent detection, optimize scanner settings

What are the key considerations for designing co-localization studies with ESF1 antibody?

Designing rigorous co-localization studies with ESF1 antibody requires attention to multiple technical parameters:

These methodological considerations ensure reliable and reproducible co-localization studies with ESF1 antibody in research settings.

How should I design experiments to validate knockdown efficiency using ESF1 antibody?

Designing robust validation experiments for ESF1 knockdown requires a multi-faceted approach:

• Western blot validation protocol:

  • Sample timing: Harvest cells 72 hours post-transfection for optimal knockdown assessment

  • Loading controls: Include both housekeeping proteins (β-actin) and compartment-specific controls (nucleolar proteins)

  • Quantification method: Use densitometry with normalization to loading controls

  • Sensitivity enhancement: For low abundance detection, consider using enhanced chemiluminescence systems

• qRT-PCR complementary validation:

  • Primer design: Create exon-junction spanning primers for ESF1 mRNA

  • Reference gene selection: Validate stability of reference genes under knockdown conditions

  • Efficiency calculation: Generate standard curves to ensure 90-110% efficiency

  • Delta-delta Ct analysis: Calculate fold change relative to control samples

• Immunofluorescence visualization:

  • Single-cell analysis: Assess knockdown heterogeneity across the population

  • Co-staining with transfection markers: For transient systems without selection

  • Nuclear counterstaining: DAPI to visualize nuclear morphology changes

• Functional readouts:

  • Pre-rRNA processing: Northern blot analysis to detect changes in pre-rRNA profiles

  • Proliferation assays: Colony formation to assess growth effects

  • Apoptosis measurement: Cell cycle analysis and apoptotic markers

  • Migration potential: Wound healing assay results

• Experimental design considerations:

  • Multiple siRNA/shRNA sequences: Test at least two targeting different regions of ESF1 mRNA

  • Concentration optimization: Test siRNAs at 5nM and 15nM concentrations

  • Appropriate controls: Include non-targeting scramble controls

  • Time course analysis: Measure knockdown at multiple time points (48h, 72h, 96h)

• Rescue experiments:

  • Express siRNA-resistant ESF1 construct

  • Validate rescue construct expression by Western blot

  • Demonstrate restoration of functional phenotypes

This comprehensive validation approach ensures reliable interpretation of ESF1 knockdown phenotypes in experimental settings.

How can ESF1 antibodies be integrated into high-throughput screening approaches?

Integration of ESF1 antibodies into high-throughput screening platforms requires specialized adaptations:

• Automated immunofluorescence workflows:

  • Robotic liquid handling for consistent antibody application

  • High-content imaging systems for nucleolar morphology quantification

  • Machine learning algorithms for pattern recognition of ESF1 distribution changes

  • Multiplexed detection with cell cycle markers to account for cell-cycle dependent variations

• Reverse-phase protein array (RPPA) applications:

  • Systematic validation of ESF1 antibody specificity in RPPA format

  • Concentration optimization for linear dynamic range

  • Normalization strategies for cross-sample comparison

  • Integration with phospho-protein detection for signaling pathway analysis

• Flow cytometry adaptation:

  • Permeabilization protocol optimization for nuclear protein detection

  • Fluorophore-conjugated ESF1 antibodies for direct detection

  • Multi-parameter analysis combining with markers for cell cycle, apoptosis, and differentiation

  • Sorting strategies for isolating ESF1-high versus ESF1-low populations

• Bead-based multiplexed assays:

  • Conjugation of ESF1 antibodies to spectrally distinct beads

  • Multiplex with antibodies against other ribosome biogenesis factors

  • Validation against standard ELISA and Western blot methods

  • Sample miniaturization for limited clinical material

• Drug screening applications:

  • Monitor ESF1 expression changes in response to ribosome biogenesis inhibitors

  • Develop reporter cell lines with fluorescent tags for endogenous ESF1

  • Establish high-throughput compatible proximity ligation assays for protein interactions

  • Design ESF1 activity biosensors for functional readouts

• Clinical sample processing:

  • Tissue microarray analysis optimization

  • Automated quantification of nuclear versus nucleolar ESF1 localization

  • Correlation with patient outcome data in ER+ breast cancer cohorts

These adaptations enable integration of ESF1 antibodies into high-throughput discovery platforms while maintaining specificity and sensitivity.

What potential exists for ESF1 antibodies in therapeutic development for ER+ breast cancer?

The therapeutic potential of ESF1 antibodies in ER+ breast cancer stems from recent discoveries about ESF1's role in cancer progression:

• Target validation evidence:

  • Proteomic identification: iTRAQ analysis identified ESF1 as significantly upregulated in ER+ breast cancer compared to adjacent normal tissue

  • Functional confirmation: Knockdown studies demonstrated ESF1's role in promoting cancer cell proliferation and migration

  • Prognostic correlation: High ESF1 expression correlates with poorer patient outcomes

• Therapeutic antibody engineering considerations:

  • Epitope selection: Target functional domains critical for ESF1's oncogenic activity

  • Internalization capacity: Evaluate antibody-drug conjugate potential

  • Format optimization: Compare conventional IgG, Fab fragments, and engineered variants

  • Affinity maturation: Enhance binding while maintaining specificity

• Delivery challenges and solutions:

  • Nuclear localization barrier: Develop cell-penetrating antibody conjugates

  • Tumor penetration strategies: Size optimization and binding site barrier evaluation

  • Combination with agents disrupting nuclear membrane integrity during mitosis

• Functional blocking strategies:

  • Protein-protein interaction disruption: Target interfaces with key ribosome biogenesis factors

  • Conformational locking: Stabilize inactive conformations

  • Nucleolar localization disruption: Prevent recruitment to pre-ribosomal complexes

• Translational research roadmap:

  • Patient-derived xenograft models: Test antibody efficacy in maintaining tissue architecture

  • Combination strategies: Evaluate synergy with existing therapies (endocrine, CDK4/6 inhibitors)

  • Resistance mechanism identification: Monitor ESF1 expression changes during treatment failure

  • Biomarker development: Identify patient subpopulations most likely to respond

• Safety considerations:

  • Differential expression quantification between normal and malignant tissue

  • Assessment of essential functions in healthy cells

  • Development of conditional targeting strategies to minimize off-target effects

While direct therapeutic targeting presents challenges due to nuclear localization, understanding ESF1 biology through antibody-based research may reveal downstream vulnerabilities that are more accessible for therapeutic intervention.

How do polyclonal versus monoclonal ESF1 antibodies compare in different research applications?

This comprehensive comparison provides researchers with selection criteria based on specific experimental requirements and research objectives.

What are the critical factors in selecting between different ESF1 antibody epitopes for specific research questions?

This epitope selection guide enables researchers to choose optimal antibodies based on specific research questions, maximizing experimental success and data interpretation accuracy.

How might ESF1 antibodies contribute to understanding the relationship between ribosome biogenesis and cancer progression?

ESF1 antibodies offer unique opportunities to investigate the mechanistic connections between ribosome biogenesis dysregulation and cancer development:

• Cancer-specific nucleolar alterations:

  • Comparative ESF1 localization patterns between normal and malignant cells

  • Correlation between nucleolar morphology changes and ESF1 distribution

  • Dynamic tracking of ESF1 during cancer progression stages

• Integration with cancer signaling networks:

  • Monitoring ESF1 response to oncogenic signaling pathway activation

  • Investigating ESF1 status following mTOR inhibition and other ribosome biogenesis targeting approaches

  • Examining ESF1 regulation by estrogen receptor signaling in ER+ breast cancer

• Therapeutic targeting strategies:

  • Identifying synthetic lethality partners for ESF1 inhibition

  • Developing ESF1-targeted proteolysis targeting chimeras (PROTACs)

  • Exploiting nucleolar stress responses induced by ESF1 disruption

• Clinical translation potential:

  • Development of ESF1 immunohistochemistry protocols for patient stratification

  • Correlation of ESF1 status with treatment response in clinical cohorts

  • Combination therapies targeting both ESF1 and traditional cancer pathways

• Multi-omic integration approaches:

  • Correlating ESF1 protein levels with ribosome profiling data

  • Integrating ESF1 status with RNA-seq and proteomics in patient samples

  • Network analysis positioning ESF1 within cancer-specific protein interaction networks

• Evolving methodologies:

  • Super-resolution microscopy for nucleolar nanostructure organization

  • Live-cell imaging with ESF1 biosensors for real-time dynamics

  • Cryo-EM structural studies of ESF1-containing complexes

These research directions leverage ESF1 antibodies to bridge fundamental ribosome biology with translational cancer research, potentially revealing novel therapeutic vulnerabilities.

What are the recommended immunofluorescence protocols for optimal ESF1 detection in different cell types?

Washing Protocol for All Methods: 3× 5 minutes in PBS between all steps

Controls:

• Primary antibody omission

• Blocking peptide competition

• Known positive control cells/tissues

• ESF1 knockdown negative control

This comprehensive protocol table provides optimized conditions for various experimental systems while highlighting critical considerations for successful ESF1 detection.

What is the optimal protocol for using ESF1 antibody in protein complex isolation and characterization?

Protocol: ESF1 Antibody-Based Protein Complex Isolation and Characterization

Cell Preparation and Lysis

• Cell culture:

  • Grow cells to 70-80% confluence in appropriate media

  • For nucleolar enrichment, grow cells in nucleolar stress-free conditions

• Cross-linking (optional):

  • Wash cells twice with ice-cold PBS

  • Add 1% formaldehyde in PBS for 10 minutes at room temperature

  • Quench with 125 mM glycine for 5 minutes

  • Wash three times with ice-cold PBS

• Cell lysis:

  • Scrape cells in ice-cold PBS containing protease inhibitors

  • Centrifuge at 500×g for 5 minutes at 4°C

  • Resuspend pellet in nuclear isolation buffer (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM DTT, protease inhibitors)

  • Add Triton X-100 to 0.1% final concentration

  • Incubate on ice for 8 minutes

  • Centrifuge at 1,300×g for 5 minutes at 4°C to pellet nuclei

• Nucleolar extraction:

  • Resuspend nuclear pellet in nucleolar lysis buffer (10 mM HEPES pH 7.9, 150 mM NaCl, 1.5 mM MgCl₂, 0.5% NP-40, 1 mM DTT, protease inhibitors)

  • Sonicate briefly (3× 10 seconds, 30% amplitude)

  • Centrifuge at 16,000×g for 10 minutes at 4°C

  • Transfer supernatant to new tube

Immunoprecipitation

• Antibody coupling to beads:

  • Wash 50 μL Protein G magnetic beads 3× with PBS-T

  • Add 5 μg rabbit anti-ESF1 antibody in 200 μL PBS-T

  • Rotate for 1 hour at room temperature

  • Wash 3× with PBS-T to remove unbound antibody

• Pre-clearing lysate (optional):

  • Add 50 μL control IgG-coupled beads to nucleolar extract

  • Rotate for 1 hour at 4°C

  • Remove beads magnetically, transfer supernatant to new tube

• Immunoprecipitation:

  • Add pre-cleared lysate to ESF1 antibody-coupled beads

  • Rotate overnight at 4°C

  • Wash 5× with wash buffer (10 mM HEPES pH 7.9, 150 mM NaCl, 1.5 mM MgCl₂, 0.1% NP-40, 1 mM DTT)

  • Include RNase inhibitors if RNA interactions are being studied

• Elution options:

  • Native: Incubate with immunizing peptide (2 mg/mL) for 2 hours at 4°C

  • Denaturing: Boil in 1× Laemmli buffer for 5 minutes

  • For cross-linked samples: Incubate at 65°C overnight in reverse cross-linking buffer

Complex Characterization

• Mass spectrometry sample preparation:

  • Separate proteins by SDS-PAGE

  • Cut gel into 1 mm slices

  • Perform in-gel trypsin digestion

  • Extract peptides for LC-MS/MS analysis

• RNA analysis (if applicable):

  • Extract RNA from beads using TRIzol reagent

  • Perform RT-PCR or RNA-seq to identify bound RNA species

  • For pre-rRNA processing analysis, use specific primers targeting processing sites

• Validation by Western blot:

  • Run 10% of IP sample on SDS-PAGE

  • Transfer to PVDF membrane

  • Probe with antibodies against suspected interacting partners

  • Confirm ESF1 pull-down efficiency

• Functional reconstitution (advanced):

  • Elute complexes under native conditions

  • Test in in vitro pre-rRNA processing assays

  • Assess complex integrity by native PAGE