ESFL2 Antibody

What is ESFL2 protein in Arabidopsis thaliana and what cellular functions does it perform?

ESFL2 (Establishment Factor-Like 2) is a protein found in Arabidopsis thaliana that has been investigated using specific antibodies such as the rabbit polyclonal antibody orb787865. While the complete functional characterization of ESFL2 is still developing in the scientific literature, it appears to be involved in plant cellular regulation processes . Unlike human proteins with similar nomenclature (such as ESCO2, which functions as an acetyltransferase in sister chromatid cohesion ), plant ESFL2 has distinct roles in Arabidopsis thaliana cellular function. Current research methodologies using ESFL2 antibodies focus on characterizing its expression patterns, localization, and potential interaction partners.

How are ESFL2 antibodies validated for specificity in plant research?

Validation of ESFL2 antibodies requires multiple complementary approaches:

• Western blot validation: Comparing wild-type Arabidopsis samples with ESFL2 knockout/knockdown lines to confirm specific band detection at the expected molecular weight

• Immunoprecipitation followed by mass spectrometry: To confirm the antibody is capturing the intended target

• Peptide competition assays: Pre-incubating the antibody with purified recombinant ESFL2 protein before application to verify signal reduction

• Cross-reactivity testing: Evaluating potential cross-reactivity with closely related proteins

Antibodies like orb787865 are developed using recombinant Arabidopsis thaliana ESFL2 protein as the immunogen , which enhances specificity. Researchers should document all validation steps in publications to support reproducibility.

What experimental applications are supported by current ESFL2 antibodies?

Based on available information, ESFL2 antibodies have been validated for several experimental applications:

Researchers should optimize these dilutions for their specific experimental conditions and plant tissue types . The antibody appears to be particularly useful for detecting recombinant or endogenous ESFL2 protein in plant extracts.

What is the optimal protocol for using ESFL2 antibody in Western blot applications?

When using ESFL2 antibody for Western blot applications, the following optimized protocol is recommended:

• Sample preparation:

  • Extract plant proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail

  • For membrane-associated proteins, consider using specialized extraction buffers with appropriate detergents

  • Quantify protein concentration using Bradford or BCA assay

• Electrophoresis and transfer:

  • Resolve 20-50 μg of total protein on 10-12% SDS-PAGE

  • Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer

• Antibody incubation:

  • Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with ESFL2 antibody (orb787865) at 1:1000 dilution in blocking buffer overnight at 4°C

  • Wash 3x with TBST, 10 minutes each

  • Incubate with HRP-conjugated secondary antibody (anti-rabbit) at 1:3000-1:5000 for 1 hour at room temperature

  • Wash 3x with TBST, 10 minutes each

• Detection:

  • Develop using ECL substrate

  • Expected band size should be verified based on the predicted molecular weight of ESFL2 in Arabidopsis thaliana

• Controls:

  • Include positive control (tissue known to express ESFL2)

  • Include negative control (ESFL2 knockout tissue if available)

  • Consider peptide competition control to verify specificity

How should researchers design co-immunoprecipitation experiments using ESFL2 antibody?

Co-immunoprecipitation (Co-IP) with ESFL2 antibody requires careful experimental design:

• Buffer optimization:

  • Use mild lysis conditions to preserve protein-protein interactions (e.g., 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 5% glycerol, protease inhibitors)

  • Avoid harsh detergents that might disrupt protein complexes

• Antibody coupling:

  • Couple ESFL2 antibody (5-10 μg) to Protein A/G beads or magnetic beads

  • Alternatively, use pre-coupled anti-rabbit IgG beads and add ESFL2 antibody separately

• Immunoprecipitation procedure:

  • Pre-clear lysate with beads lacking antibody to reduce non-specific binding

  • Incubate cleared lysate with antibody-coupled beads for 2-4 hours at 4°C

  • Wash 4-5 times with buffer containing reduced detergent concentration

  • Elute protein complexes with SDS sample buffer or low pH buffer

• Analysis:

  • Analyze immunoprecipitated proteins by Western blot for known/suspected interacting partners

  • Consider mass spectrometry for unbiased identification of interaction partners

  • Verify interactions with reciprocal Co-IP or alternative methods (e.g., Y2H, BiFC)

• Controls:

  • Include IgG control (same species as ESFL2 antibody)

  • Include input sample (pre-IP lysate)

  • Consider RNase/DNase treatment to exclude nucleic acid-mediated interactions

What considerations are important when using ESFL2 antibody for immunolocalization studies?

When performing immunolocalization with ESFL2 antibody, consider these critical factors:

• Tissue preparation:

  • For immunohistochemistry: Fix tissue in 4% paraformaldehyde, embed in paraffin or resin, section at 5-10 μm

  • For immunofluorescence: Fix in 4% paraformaldehyde, prepare thin sections or use whole-mount techniques for smaller tissues

• Antigen retrieval:

  • Heat-mediated: Citrate buffer (pH 6.0), 95°C for 10-20 minutes

  • Enzymatic: Proteinase K (1-10 μg/ml) for 5-15 minutes

• Antibody incubation:

  • Block with 5% normal serum, 0.3% Triton X-100 in PBS for 1-2 hours

  • Incubate with ESFL2 antibody at 1:100-1:500 dilution overnight at 4°C

  • Use fluorescent or enzymatic (HRP/AP) secondary antibodies as appropriate

• Controls:

  • Omit primary antibody (secondary antibody only)

  • Use pre-immune serum

  • Include tissue from ESFL2 knockdown/knockout plants

  • Peptide competition control

• Counterstaining:

  • DAPI for nuclear visualization

  • Cell wall stains (e.g., calcofluor white) for plant cell architecture

• Analysis:

  • Use confocal microscopy for high-resolution subcellular localization

  • Perform colocalization studies with organelle markers to determine precise subcellular distribution

How can researchers address weak or absent signals when using ESFL2 antibody in Western blots?

When encountering weak or absent signals with ESFL2 antibody in Western blots, consider this systematic approach:

• Sample preparation issues:

  • Increase protein loading (50-100 μg total protein)

  • Verify sample integrity (run Coomassie-stained gel in parallel)

  • Use fresh tissue extraction with enhanced protease inhibitors

  • Modify extraction buffer (try different detergents, salt concentrations)

• Antibody conditions:

  • Increase antibody concentration (try 1:500 or 1:250 dilution)

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

  • Test different blocking agents (BSA vs. non-fat milk)

  • Try enhanced sensitivity detection systems (femto ECL substrates)

• Technical considerations:

  • Verify transfer efficiency (use stained markers or reversible membrane staining)

  • Reduce washing stringency (shorter washes, lower detergent concentration)

  • Use fresh antibody aliquot (avoid repeated freeze-thaw cycles)

  • Try alternative membrane type (PVDF vs. nitrocellulose)

• Protein detectability factors:

  • Consider protein expression levels and timing (use tissues/conditions with highest expression)

  • Account for post-translational modifications that might affect epitope recognition

  • Check if denaturing conditions affect epitope (try native conditions if appropriate)

• Analysis approach:

  • Use positive control (recombinant ESFL2 protein if available)

  • Consider tissue-specific expression patterns from transcriptomic data to select appropriate samples

  • Try membrane stripping and reprobing if other antibodies worked on the same membrane

What strategies help distinguish specific from non-specific binding with ESFL2 antibody?

To distinguish between specific and non-specific binding when using ESFL2 antibody:

• Experimental controls:

  • Use genetic controls (knockout/knockdown lines) as gold standard

  • Perform peptide competition assay by pre-incubating antibody with immunizing peptide/protein

  • Compare signal patterns across different tissues with known expression patterns

• Analytical approaches:

  • Evaluate band pattern - specific bands should appear at consistent molecular weights

  • Non-specific bands often vary in intensity across samples independenty of target protein

  • Specific signal should correlate with expression data from other methods

• Validation techniques:

  • Analyze subcellular fractionation to confirm signal in expected compartment

  • Use alternative antibodies raised against different epitopes

  • Confirm with orthogonal techniques (e.g., mass spectrometry)

• Quantitative assessment:

  • Calculate signal-to-noise ratio across multiple experiments

  • Perform densitometry on bands of interest relative to background

Maintaining detailed records of all binding patterns across different experimental conditions can help build confidence in identifying truly specific signals .

How should researchers quantify and statistically analyze Western blot data using ESFL2 antibody?

Proper quantification and statistical analysis of Western blot data with ESFL2 antibody requires rigorous methodology:

• Image acquisition:

  • Capture images within linear dynamic range of detection

  • Avoid saturated pixels that will underestimate differences

  • Include standards for calibration if possible

• Quantification procedure:

  • Use software like ImageJ, Image Lab, or similar tools for densitometry

  • Subtract local background for each lane

  • Normalize to appropriate loading controls (e.g., GAPDH, actin, tubulin)

  • Consider total protein normalization methods (e.g., stain-free technology, Ponceau S)

• Experimental design for statistical validity:

  • Perform at least 3 independent biological replicates

  • Use technical replicates to assess measurement variance

  • Design balanced experiments with appropriate controls

• Statistical approaches:

  • Use parametric tests (t-test, ANOVA) if normality assumptions are met

  • Consider non-parametric alternatives if data distribution is skewed

  • Apply multiple testing corrections for comparisons across many samples

  • Calculate confidence intervals around fold changes

• Data presentation:

  • Present both representative blot images and quantitative graphs

  • Include error bars representing standard deviation or standard error

  • Indicate statistical significance using appropriate notation

  • Report exact p-values rather than threshold ranges

• Methodological considerations:

  • Verify antibody linearity range through dilution series

  • Account for normalization control stability across experimental conditions

  • Consider using housekeeping protein index (combination of multiple controls)

How can ESFL2 antibody be used for chromatin immunoprecipitation (ChIP) studies?

While ESFL2 is not typically characterized as a DNA-binding protein, ChIP could be used to investigate whether it associates with chromatin indirectly through protein complexes. This advanced application requires:

• Protocol optimization:

  • Cross-link plant tissue with 1-3% formaldehyde for 10-15 minutes

  • Use appropriate chromatin shearing method (sonication or enzymatic digestion)

  • Optimize shearing to achieve fragments of 200-500 bp

  • Perform immunoprecipitation with 5-10 μg ESFL2 antibody

• Controls and validation:

  • Include IgG control from same species as ESFL2 antibody

  • Use positive control antibody (e.g., against histone H3)

  • Perform ChIP in ESFL2 knockout/knockdown plants as negative control

  • Validate enrichment by qPCR before proceeding to sequencing

• Analysis approach:

  • For ChIP-qPCR: Design primers for candidate regions and control regions

  • For ChIP-seq: Prepare libraries according to standard protocols

  • Analyze data using specialized software (MACS2, Homer)

  • Integrate with gene expression data to identify functional correlations

• Interpretation considerations:

  • If ESFL2 is not a DNA-binding protein, enrichment patterns may reflect indirect associations

  • Compare binding profiles with known transcription factors or chromatin modifiers

  • Consider sequential ChIP (re-ChIP) to identify co-binding with interaction partners

What approaches should be used to study post-translational modifications of ESFL2 protein?

Investigating post-translational modifications (PTMs) of ESFL2 requires specialized techniques:

• Identification strategies:

  • Immunoprecipitate ESFL2 using validated antibody

  • Analyze by mass spectrometry (LC-MS/MS) with PTM-specific methods

  • Use phospho-enrichment techniques (TiO2, IMAC) for phosphorylation studies

  • Apply targeted approaches for specific modifications (ubiquitination, acetylation)

• Verification methods:

  • Generate or obtain modification-specific antibodies

  • Use Phos-tag SDS-PAGE for phosphorylation analysis

  • Employ 2D gel electrophoresis to separate modified forms

  • Apply specific inhibitors or enhancers of modification enzymes

• Functional characterization:

  • Create site-directed mutants of modified residues

  • Express wildtype and mutant forms in plant systems

  • Conduct complementation studies in knockouts

  • Analyze protein interaction profiles and subcellular localization

• Experimental design:

  • Include appropriate controls for each modification type

  • Study modifications across developmental stages and stress conditions

  • Consider temporal dynamics using pulse-chase experiments

  • Integrate with proteomic datasets of related proteins

• Analysis workflow:

  • Map modifications to protein domains and motifs

  • Use bioinformatic tools to predict functional consequences

  • Compare conservation of modification sites across species

  • Build modification networks through pathway analysis

How can researchers develop custom ESFL2 antibodies for specific epitopes?

For researchers requiring antibodies targeting specific regions or forms of ESFL2, custom antibody development involves:

• Epitope selection strategy:

  • Analyze protein sequence for antigenic regions using prediction algorithms

  • Consider accessibility of epitopes in native protein

  • Target unique regions that distinguish ESFL2 from related proteins

  • Select regions based on research questions (e.g., domains, modification sites)

• Immunization approaches:

  • Peptide antigens (15-25 amino acids) for site-specific antibodies

  • Recombinant protein fragments for domain-specific antibodies

  • Full-length protein for maximum epitope coverage

  • Consider carrier proteins and adjuvants appropriate for host species

• Production considerations:

  • Select between polyclonal (rapid, multiple epitopes) or monoclonal (single epitope, renewable)

  • Choose appropriate host species (rabbit, mouse, chicken, etc.)

  • For monoclonals, plan hybridoma screening strategy

  • Consider synthetic antibody technologies (phage display, yeast display)

• Validation requirements:

  • Test against recombinant protein and native extracts

  • Perform peptide competition assays

  • Validate in knockout/knockdown systems

  • Cross-validate with mass spectrometry

• Advanced modifications:

  • Engineer recombinant antibodies for enhanced properties

  • Consider fragment antibodies (Fab, scFv) for specialized applications

  • Develop conjugated antibodies for specific detection methods

  • Create modification-specific antibodies using modified peptides as immunogens

What emerging single-cell techniques can be applied using ESFL2 antibody?

Emerging single-cell techniques that could be adapted for ESFL2 research include:

• Single-cell proteomics approaches:

  • Mass cytometry (CyTOF) with metal-conjugated ESFL2 antibodies

  • Single-cell Western blotting using microfluidic platforms

  • Proximity ligation assay (PLA) for protein interaction studies at single-cell level

  • Microfluidic antibody capture for quantitative single-cell protein analysis

• Spatial profiling technologies:

  • Imaging mass cytometry for tissue sections

  • Multiplexed ion beam imaging (MIBI) for high-parameter spatial analysis

  • CO-Detection by indEXing (CODEX) for highly multiplexed tissue imaging

  • Spatial transcriptomics integrated with protein detection

• Live-cell applications:

  • Intrabodies derived from ESFL2 antibodies for live-cell imaging

  • Split-fluorescent protein complementation with antibody fragments

  • Optogenetic control of protein function using antibody-based tools

  • Nanobody development for enhanced penetration and reduced interference

• Integration with other single-cell data:

  • CITE-seq-like approaches for simultaneous protein and RNA profiling

  • Multi-modal single-cell analysis platforms

  • Computational integration of protein and transcriptome data

  • Trajectory analysis incorporating protein dynamics

• Technical considerations:

  • Antibody specificity becomes even more critical at single-cell resolution

  • Signal amplification may be necessary for low-abundance proteins

  • Cell isolation and preservation methods affect protein epitope integrity

  • Quantitative standards should be incorporated for absolute quantification

How does ESFL2 protein research compare with investigations of related proteins in plants?

Contextualizing ESFL2 research within the broader landscape of plant protein studies:

• Comparative analysis approach:

  • Compare with other establishment factor-like proteins in Arabidopsis

  • Examine functional conservation across plant species

  • Contrast with analogous proteins in non-plant systems

  • Consider evolutionary relationships and functional divergence

• Research methodology comparison:

  • Apply techniques successful with related proteins

  • Adapt antibody-based protocols from other plant protein studies

  • Consider lessons learned from challenges with similar proteins

  • Evaluate whether different epitope selection strategies were successful

• Functional context:

  • Compare expression patterns and regulation mechanisms

  • Assess involvement in similar biological processes

  • Examine whether interaction partners overlap

  • Consider redundancy and specialization within protein families

• Technical challenges:

  • Compare antibody generation success rates for related proteins

  • Identify common technical hurdles in plant protein studies

  • Evaluate whether similar epitopes proved immunogenic

  • Consider epitope masking in different cellular contexts

• Research progress assessment:

  • Map the current state of ESFL2 knowledge relative to related proteins

  • Identify knowledge gaps that might benefit from comparative approaches

  • Consider collaborative opportunities with researchers studying related proteins

  • Evaluate whether techniques from other fields could advance ESFL2 research

How can ESFL2 antibody data be integrated with other -omics approaches?

Integrating ESFL2 antibody-based research with complementary -omics approaches:

• Multi-omics integration strategy:

  • Correlate protein expression data with transcriptomics

  • Integrate with phosphoproteomics and other PTM studies

  • Connect with metabolomic changes in ESFL2 mutants

  • Incorporate information from protein interaction networks

• Experimental design for integration:

  • Plan coordinated sampling for multiple -omics analyses

  • Include appropriate controls and standards for cross-platform normalization

  • Consider time-course studies to capture dynamic relationships

  • Use appropriate statistical methods for multi-omics data integration

• Analytical approaches:

  • Apply dimension reduction techniques (PCA, t-SNE) to identify patterns

  • Use network analysis to map relationships between different data types

  • Perform pathway enrichment across multiple data layers

  • Consider machine learning approaches for integrative pattern recognition

• Validation strategy:

  • Design targeted experiments to test hypotheses generated by integrative analysis

  • Use ESFL2 antibody-based techniques to validate specific predictions

  • Consider genetic approaches (CRISPR, RNAi) to test functional relationships

  • Develop mathematical models to explain multi-level regulation

• Resources and tools:

  • Utilize specialized software for multi-omics data integration

  • Leverage plant-specific databases for functional annotation

  • Consider public data repositories for comparative analysis

  • Employ visualization tools designed for complex data relationships