ERF1B Antibody

What is ERF1B and how does it function in plant signaling pathways?

ERF1B belongs to the ERF (Ethylene Response Factor) family of transcription factors involved in plant signaling networks. Based on molecular research, ERF1B expression is upregulated in ebp1 mutants, suggesting a potential regulatory relationship between EBP1 and ERF1B . ERF1B likely functions as a transcription factor that regulates gene expression in response to various signaling cascades, potentially including the RALF1-FER pathway.

The methodological approach to studying ERF1B function typically involves:

• Gene expression analysis via qRT-PCR to quantify ERF1B levels in different genetic backgrounds

• Chromatin immunoprecipitation (ChIP) using validated ERF1B antibodies to identify DNA binding sites

• Transcriptome analysis of ERF1B overexpression or knockout lines to identify downstream targets

• Protein-protein interaction studies to place ERF1B within signaling complexes

How do I select the appropriate ERF1B antibody for my specific research application?

When selecting an ERF1B antibody, researchers should consider several methodological factors:

• Antibody type (polyclonal vs. monoclonal):

  • Polyclonal antibodies offer broader epitope recognition but potentially lower specificity

  • Monoclonal antibodies provide higher specificity but may be affected by epitope masking

• Application compatibility:

  • For western blotting: Antibodies recognizing denatured epitopes

  • For immunoprecipitation: Antibodies recognizing native conformations

  • For immunofluorescence: Validated antibodies with minimal background binding

• Validation status:

  • Knockout/knockdown controls demonstrating specificity

  • Cross-reactivity testing with related ERF family members

  • Published research employing the antibody in similar applications

• Species reactivity:

  • Confirm the antibody recognizes ERF1B from your model organism

  • Consider sequence homology when working with non-model species

What are the relationships between ERF1B and other components of the RALF1-FER signaling pathway?

Based on current research, ERF1B appears to be regulated downstream of the EBP1 pathway . The connection can be summarized as:

• RALF1 peptide binds to FERONIA (FER) receptor kinase at the cell membrane

• FER interacts with and potentially phosphorylates EBP1

• EBP1 accumulates in the nucleus upon RALF1 treatment

• EBP1 regulates the expression of various genes, including ERF1B

To study these relationships methodologically:

• Co-immunoprecipitation using anti-ERF1B antibodies can identify physical interactions with other signaling components

• ChIP-seq analysis can identify if EBP1 directly binds to the ERF1B promoter

• Genetic studies combining mutations in multiple pathway components (e.g., fer-4 with erf1b) can reveal epistatic relationships

What are the optimal sample preparation methods for ERF1B detection in different experimental contexts?

Sample preparation significantly impacts ERF1B antibody performance across different applications:

For Western Blotting:

• Extraction buffer optimization:

  • RIPA buffer (with protease inhibitors) for general protein extraction

  • Nuclear extraction protocols for enriched nuclear fraction where ERF1B likely functions as a transcription factor

  • Consider including phosphatase inhibitors to preserve potential post-translational modifications

• Protein handling:

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

  • Use freshly prepared samples when possible, as freeze-thaw cycles may affect epitope integrity

For Immunoprecipitation:

• Crosslinking considerations:

  • Reversible crosslinkers like DSP (dithiobis(succinimidyl propionate)) can help stabilize transient interactions

  • Formaldehyde (1%) crosslinking for 10 minutes is suitable for chromatin immunoprecipitation

For Immunofluorescence:

• Fixation methods:

  • 4% paraformaldehyde for 15-20 minutes preserves most epitopes

  • Methanol fixation (-20°C) may better preserve certain nuclear antigens

  • Include permeabilization step with 0.1% Triton X-100 for nuclear factors

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) may improve detection of nuclear factors like ERF1B

How can ERF1B antibodies be effectively used in protein-protein interaction studies?

ERF1B antibodies can elucidate protein-protein interactions through several methodological approaches:

Co-Immunoprecipitation (Co-IP):

• Design protocol similar to how FER-EBP1 interactions were studied :

  • Prepare total protein extracts from plant tissues under native conditions

  • Pre-clear lysates with protein A/G beads

  • Incubate with ERF1B antibody (4°C, overnight)

  • Capture immunocomplexes with fresh protein A/G beads

  • Wash extensively to remove non-specific interactions

  • Elute and analyze by western blotting for potential interaction partners

Proximity Ligation Assay (PLA):

• This technique detects protein interactions in situ:

  • Fix and permeabilize tissue samples

  • Incubate with primary antibodies against ERF1B and suspected interaction partner

  • Apply PLA probes with complementary oligonucleotides

  • Ligation and amplification steps

  • Visualization of discrete spots indicating <40nm proximity of proteins

Bimolecular Fluorescence Complementation (BiFC):

• Following approaches used for FER protein interactions :

  • Create fusion constructs of ERF1B with one half of YFP

  • Fuse potential interaction partners with complementary YFP fragment

  • Co-express in protoplasts or plant tissues

  • Observe reconstituted fluorescence indicating interaction

  • Validate expression of fusion proteins by western blot

What controls should be included when using ERF1B antibodies in immunolocalization studies?

Proper controls are essential for reliable ERF1B immunolocalization:

Negative Controls:

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

• Isotype control - Use non-relevant antibody of same isotype/host species

• Genetic controls - Include ERF1B knockout/knockdown samples when available

• Peptide competition - Pre-incubate antibody with immunizing peptide to block specific binding

Positive Controls:

• Samples with known ERF1B overexpression

• Positive reference tissues with validated ERF1B expression

• Parallel detection of known nuclear markers when examining ERF1B nuclear localization

Internal Controls:

• Co-staining with organelle markers to confirm subcellular localization:

  • DAPI for nuclear localization

  • Membrane markers if examining potential membrane association

• Signal quantification controls:

  • Fixed exposure settings across all samples

  • Include calibration standards for intensity measurements

How can I improve ERF1B antibody specificity in complex plant tissue samples?

Enhancing specificity requires methodological refinements:

Pre-adsorption Protocol:

• Incubate antibody with plant extract from ERF1B knockout/knockdown tissue

• Remove antibodies binding to non-specific proteins

• Use pre-cleared antibody solution for your experiment

Optimized Blocking:

• Test alternative blocking agents:

  • 5% non-fat dry milk in TBS-T

  • 5% BSA for phospho-specific applications

  • Commercial blocking solutions optimized for plant samples

• Extend blocking time to 2 hours at room temperature or overnight at 4°C

Modified Antibody Incubation:

• Dilution series testing (1:500 to 1:5000) to identify optimal concentration

• Reduce temperature to 4°C and extend incubation time to overnight

• Add 0.05% Tween-20 to antibody dilution buffer to reduce non-specific binding

Washing Optimization:

• Increase number of washes (5-6 times, 10 minutes each)

• Use higher salt concentration in wash buffer (up to 500mM NaCl)

• Add 0.1% SDS to washing buffer for western blot applications

What are common causes of inconsistent ERF1B staining patterns in immunofluorescence?

Several methodological factors can cause inconsistent immunofluorescence results:

Fixation Variables:

• Overfixation - Excessive crosslinking can mask epitopes

  • Solution: Reduce fixation time or concentration

  • Try alternative fixatives (methanol vs. paraformaldehyde)

Antibody Penetration Issues:

• Insufficient permeabilization

  • Solution: Increase Triton X-100 concentration (0.2-0.5%)

  • Consider detergent treatment duration (15-30 minutes)

Epitope Masking:

• Protein-protein interactions hiding ERF1B epitopes

  • Solution: Test different antigen retrieval methods:

    • Heat-induced epitope retrieval (95°C, 20 minutes in citrate buffer)

    • Enzymatic retrieval with proteinase K (very mild conditions)

Signal Variability:

• Heterogeneous ERF1B expression or localization

  • Solution: Standardize experimental conditions

    • Synchronize cell populations if possible

    • Control treatment timing precisely, as ERF1B may show dynamic localization like EBP1

How do I validate ERF1B antibody specificity in my experimental system?

Methodological approach to antibody validation:

Genetic Validation:

• Test antibody on ERF1B knockout/knockdown samples

• Expected outcome: Reduced or absent signal compared to wild-type

Molecular Weight Confirmation:

• For western blotting:

  • Verify single band at predicted molecular weight

  • Test recombinant ERF1B protein as positive control

Peptide Competition:

• Pre-incubate antibody with immunizing peptide

• Apply to identical samples in parallel

• Expected outcome: Blocked antibody should show minimal signal

Orthogonal Detection Methods:

• Compare antibody results with:

  • ERF1B-GFP fusion protein localization

  • Mass spectrometry identification of immunoprecipitated proteins

  • RNA expression data for correlation with protein signal

How can ERF1B antibodies be used in chromatin immunoprecipitation studies?

ChIP methodology for ERF1B studies:

ChIP Protocol Optimization:

• Crosslinking conditions:

  • 1% formaldehyde, 10 minutes at room temperature

  • Quench with 125mM glycine

  • For potentially weak interactions, consider using dual crosslinkers (formaldehyde + DSG)

• Chromatin fragmentation:

  • Sonication parameters: 10-15 cycles (30s ON/30s OFF)

  • Target fragment size: 200-500bp

  • Verify fragmentation by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate with ERF1B antibody overnight at 4°C

  • Include IgG negative control and positive control (e.g., RNA Pol II antibody)

  • Wash stringently to remove non-specific binding

• Analysis options:

  • ChIP-qPCR for candidate target genes

  • ChIP-seq for genome-wide binding profile

  • CUT&RUN as alternative with potentially lower background

Data Analysis Considerations:

• Peak calling algorithms (MACS2) with appropriate parameters

• Motif discovery analysis to identify ERF1B binding consensus

• Integration with RNA-seq data to correlate binding with expression changes

• Comparison with other transcription factor binding profiles

What approaches can be used to study ERF1B post-translational modifications?

Studying ERF1B post-translational modifications requires specialized approaches:

Phosphorylation Analysis:

• Phospho-specific antibody development:

  • Identify potential phosphorylation sites through bioinformatics

  • Generate phospho-specific antibodies against predicted sites

  • Validate using phosphatase treatment controls

• Mass spectrometry approaches:

  • Immunoprecipitate ERF1B using validated antibodies

  • Enrich for phosphopeptides using TiO₂ or IMAC

  • Analyze by LC-MS/MS to identify modification sites

  • Compare modification patterns before/after signaling activation

Other PTM Analyses:

• For ubiquitination:

  • Co-IP under denaturing conditions with ERF1B antibody

  • Probe with anti-ubiquitin antibodies

  • Use proteasome inhibitors (MG132) to stabilize ubiquitinated species

• For SUMOylation:

  • Similar to ubiquitination analysis

  • Use SUMO-specific antibodies

  • Include SUMO protease inhibitors (N-ethylmaleimide)

How can ERF1B antibodies help elucidate transcriptional regulation networks?

Antibody-based approaches to map ERF1B regulatory networks:

Sequential ChIP (Re-ChIP):

• Methodology for identifying co-binding transcription factors:

  • Perform standard ChIP with ERF1B antibody

  • Elute complexes under non-denaturing conditions

  • Perform second round of ChIP with antibody against potential partner

  • Analyze enriched regions shared by both factors

ChIP-MS:

• Identify chromatin-associated ERF1B interactors:

  • Perform ChIP with ERF1B antibody

  • Analyze immunoprecipitated material by mass spectrometry

  • Identify proteins co-enriched with ERF1B on chromatin

Integrative Network Analysis:

• Combine multiple data types:

  • ERF1B ChIP-seq for DNA binding sites

  • RNA-seq of ERF1B perturbation for expression changes

  • Protein interaction data from Co-IP/MS

  • Generate network models integrating these datasets

How can I combine ERF1B antibody studies with transcriptomics to identify direct target genes?

Integrated methodologies to connect ERF1B binding with gene regulation:

ChIP-seq with RNA-seq Integration:

• Experimental design:

  • Perform ChIP-seq with ERF1B antibody to identify genome-wide binding sites

  • Conduct parallel RNA-seq on:

    • ERF1B knockout/knockdown

    • ERF1B overexpression

    • Wild-type controls

  • Include time-course analysis after stimulus (e.g., RALF1 treatment) to capture dynamic regulation

• Analytical workflow:

  • Identify differentially expressed genes (DEGs) from RNA-seq

  • Map ERF1B binding sites relative to gene structures

  • Determine overlap between ERF1B-bound genes and DEGs

  • Classify direct targets (bound + differentially expressed) vs. indirect targets

• Validation approaches:

  • ChIP-qPCR for selected targets

  • Reporter gene assays with wild-type and mutated ERF1B binding sites

  • CRISPR interference at ERF1B binding sites to validate functional importance

What imaging techniques are most effective for studying ERF1B localization and dynamics?

Advanced imaging approaches for ERF1B spatial and temporal analysis:

Superresolution Microscopy:

• Structured Illumination Microscopy (SIM):

  • ~120nm resolution

  • Compatible with standard fluorophores

  • Ideal for general nuclear distribution patterns of ERF1B

• Stochastic Optical Reconstruction Microscopy (STORM):

  • ~20nm resolution

  • Requires special fluorophores and buffers

  • Useful for precise subnuclear localization

• Sample preparation considerations:

  • Thin sections (≤5μm) for optimal resolution

  • High-quality primary antibodies with minimal background

  • Bright, photostable fluorophores for secondary detection

Live-Cell Imaging:

• Fluorescent protein fusions:

  • Create ERF1B-FP (fluorescent protein) fusions

  • Verify functionality through complementation assays

  • Use spinning disk confocal for rapid acquisition with minimal photobleaching

• Dynamics measurements:

  • FRAP (Fluorescence Recovery After Photobleaching) to measure ERF1B mobility

  • Single-particle tracking to follow individual ERF1B molecules

  • Optogenetic approaches to control ERF1B activity with light

How does ERF1B function compare across different plant species and experimental systems?

Comparative analysis methodology:

Cross-Species Antibody Applications:

• Epitope conservation analysis:

  • Align ERF1B sequences across species of interest

  • Identify conserved regions suitable for antibody recognition

  • Test antibody cross-reactivity on protein extracts from different species

• Experimental design considerations:

  • Include positive controls from species where antibody is known to work

  • Optimize extraction protocols for each species (tissue-specific modifications)

  • Consider using multiple antibodies targeting different epitopes

Heterologous Expression Systems:

• Recombinant ERF1B production:

  • Express ERF1B from different species in E. coli or yeast

  • Purify using affinity tags

  • Test antibody recognition of recombinant proteins

  • Compare binding properties to DNA elements

• Functional conservation testing:

  • Complementation assays across species

  • DNA binding specificity comparison through EMSA or protein binding microarrays

  • Use antibodies to perform comparative ChIP-seq across species