At4g00755 Antibody

What is the At4g00755 protein and why are antibodies against it valuable for plant research?

At4g00755 is an F-box family protein found in Arabidopsis thaliana (Mouse-ear cress), a widely used model organism in plant molecular biology . F-box proteins are components of SCF ubiquitin-ligase complexes that regulate protein degradation through the ubiquitin-proteasome pathway.

Antibodies against At4g00755 are valuable research tools because they enable:

• Detection and quantification of At4g00755 protein expression in different tissues

• Investigation of protein-protein interactions involving At4g00755

• Study of post-translational modifications

• Examination of protein localization through immunohistochemistry techniques

These applications help researchers understand the role of this F-box protein in plant development, stress responses, and cellular signaling pathways.

What validation methods should be used to confirm At4g00755 antibody specificity?

Proper validation of At4g00755 antibody specificity is crucial for reliable research results. Recommended validation methods include:

• Western blot analysis using:

  • Wild-type Arabidopsis thaliana tissue

  • At4g00755 knockout/knockdown mutants as negative controls

  • Tissues overexpressing tagged At4g00755 protein as positive controls

• Immunoprecipitation followed by mass spectrometry to confirm that the antibody indeed pulls down the At4g00755 protein

• Pre-absorption tests where the antibody is pre-incubated with the purified antigen before immunodetection

• Cross-reactivity testing against related F-box proteins to ensure specificity

• Immunohistochemistry comparisons between wild-type and knockout plants

According to available information, commercially available At4g00755 antibodies have been tested for applications such as ELISA and Western blot , but individual researchers should perform additional validation in their specific experimental contexts.

What are the recommended experimental conditions for using At4g00755 antibody in Western blot applications?

Based on validated protocols for plant F-box protein antibodies:

Sample preparation:

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

• Use fresh tissue when possible or flash-freeze in liquid nitrogen

• Include reducing agents like DTT or β-mercaptoethanol in sample buffer

Western blot conditions:

• Protein separation: 10-12% SDS-PAGE

• Transfer: Standard PVDF membrane transfer at 100V for 1 hour

• Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody: Dilute At4g00755 antibody 1:1000 in blocking solution, incubate overnight at 4°C

• Secondary antibody: Anti-rabbit HRP at 1:5000 dilution for 1 hour at room temperature

• Detection: Standard ECL detection systems

Storage and handling:

• Store antibody at -20°C or -80°C

• Avoid repeated freeze-thaw cycles

• Use 0.03% Proclin 300 as preservative in 50% glycerol and 0.01M PBS (pH 7.4)

How can At4g00755 antibody be used to investigate protein-protein interactions in histone modification pathways?

At4g00755 antibody can be leveraged to study potential interactions between this F-box protein and components of histone modification complexes like NuA4, which is involved in histone acetylation and transcriptional regulation in plants.

Methodological approach:

• Co-immunoprecipitation (Co-IP):

  • Crosslink protein complexes in plant tissues

  • Immunoprecipitate using At4g00755 antibody

  • Analyze precipitated proteins by mass spectrometry to identify interacting partners

• Chromatin Immunoprecipitation (ChIP):

  • Follow protocols similar to those used for NuA4 complex studies

  • Use spike-in controls with predetermined proportions of mouse chromatin as reference

  • Compare binding patterns with histone modification marks like H4K5ac and H3K9ac

• Proximity-dependent labeling:

  • Create fusion proteins of At4g00755 with BioID or APEX2

  • Use the antibody to validate expression of the fusion protein

  • Identify proximal proteins through biotinylation and streptavidin pulldown

Analyzing data from these experiments can reveal whether At4g00755 is involved in chromatin-mediated transcriptional regulation, potentially connecting ubiquitin-mediated protein degradation to epigenetic regulation pathways.

What approaches can be used to study the role of At4g00755 in plant stress responses using the specific antibody?

To investigate At4g00755's role in plant stress responses, researchers can employ several antibody-dependent methodologies:

Temporal and spatial expression analysis:

• Collect plant tissues at different time points after stress exposure (drought, salt, pathogen, cold)

• Perform Western blot analysis with At4g00755 antibody to track protein abundance changes

• Complement with RT-qPCR for transcript level comparison

Subcellular localization under stress:

• Use immunofluorescence with At4g00755 antibody to track protein localization changes

• Compare normal vs. stress conditions

• Co-stain with organelle markers to identify translocation events

Protein modification detection:

• Use phospho-specific antibodies alongside At4g00755 antibody

• Analyze ubiquitination status changes using immunoprecipitation with At4g00755 antibody followed by ubiquitin detection

Comparative analysis with mutant lines:

• Compare protein levels in wild-type plants versus At4g00755 decoy expressing plants

• Use antibody to validate decoy expression from pB7HFN-AT4G00755 constructs

This multi-faceted approach can reveal how At4g00755 protein levels, modifications, and localization change during stress responses, providing insights into its role in plant adaptation mechanisms.

How can epitope mapping be performed for At4g00755 antibody to determine its exact binding site?

Epitope mapping is essential for understanding antibody specificity and can aid in experimental design. For At4g00755 antibody, consider these approaches:

Peptide array analysis:

• Generate overlapping peptides (15-20 amino acids) covering the entire At4g00755 protein sequence

• Spot peptides onto membrane

• Probe with the At4g00755 antibody

• Detect binding to identify reactive peptides representing the epitope

Deletion mutant analysis:

• Express a series of truncated At4g00755 proteins with sequential deletions

• Perform Western blot with the antibody

• Identify the smallest fragment still recognized by the antibody

Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

• Compare deuterium uptake patterns of At4g00755 protein alone versus antibody-bound protein

• Regions protected from exchange when antibody is bound represent the epitope

Computational prediction validation:

• Use bioinformatic tools to predict antigenic regions of At4g00755

• Synthesize predicted epitope peptides

• Test antibody binding to these peptides

• Compare with experimental results from above methods

Understanding the exact epitope can help determine if the antibody binds to functionally important domains and whether it might interfere with protein-protein interactions in immunoprecipitation experiments.

What are the most common issues encountered when using At4g00755 antibody and how can they be resolved?

Researchers working with plant protein antibodies like At4g00755 antibody commonly encounter these challenges:

For plant-specific challenges:

• High levels of phenolic compounds and polysaccharides can interfere with protein extraction; add PVPP to extraction buffer

• Abundant RuBisCO can mask lower-abundance proteins; use fractionation techniques

• Secondary metabolites may cause non-specific interactions; pre-clear lysates with normal IgG

How should researchers optimize chromatin immunoprecipitation (ChIP) protocols for At4g00755 antibody?

Optimizing ChIP protocols for At4g00755 antibody requires careful consideration of several parameters:

Crosslinking optimization:

• Test different formaldehyde concentrations (0.5-3%)

• Optimize crosslinking time (10-30 minutes)

• Consider dual crosslinking with DSG followed by formaldehyde for protein-protein interactions

Chromatin preparation:

• Optimize sonication conditions to achieve 200-500bp fragments

• Verify fragmentation by agarose gel electrophoresis

• Include spike-in controls with predetermined proportions of mouse chromatin as reference, similar to methods used in NuA4 studies

Immunoprecipitation conditions:

• Test different antibody amounts (2-10 μg per ChIP)

• Compare various bead types (Protein A, Protein G, or magnetic beads)

• Optimize wash stringency to reduce background without losing signal

Controls to include:

• Input chromatin (pre-immunoprecipitation)

• IgG negative control

• Positive control using antibody against histone marks like H3K9ac

• At4g00755 knockout/knockdown plant material as negative control

Data analysis considerations:

• Normalize to spike-in control to account for global changes in epitope abundance

• Average signals over relevant genomic features (e.g., first 500bp of transcribed regions)

• Compare H3-normalized signals to account for differences in nucleosome occupancy

This optimized approach will help identify potential genomic targets of At4g00755, providing insights into its role in transcriptional regulation.

How can researchers distinguish between direct and indirect effects when interpreting At4g00755 antibody ChIP-seq data?

Distinguishing direct from indirect effects in At4g00755 ChIP-seq data requires sophisticated analytical approaches:

Integrative approaches:

• Motif analysis:

  • Identify enriched DNA motifs in At4g00755-bound regions

  • Compare with known transcription factor binding sites

  • De novo motif discovery may reveal novel binding preferences

• Multi-omics integration:

  • Correlate ChIP-seq peaks with RNA-seq data from At4g00755 mutants

  • Genes showing both binding and expression changes are likely direct targets

  • Apply criteria similar to those used in NuA4 studies: significant changes in expression (log2 fold change threshold) combined with antibody binding evidence

• Temporal resolution studies:

  • Perform time-course experiments after inducible expression/depletion of At4g00755

  • Early responding genes with binding sites are more likely direct targets

  • Late responding genes without binding sites suggest indirect regulation

• Validation through orthogonal techniques:

  • Use CUT&RUN or CUT&Tag for higher resolution binding data

  • Perform reporter assays with identified binding regions

  • Use CRISPR interference at binding sites to verify functional relevance

This systematic approach helps researchers distinguish between genes directly regulated by At4g00755 binding versus those affected through secondary mechanisms.

What computational analyses are recommended for processing At4g00755 antibody-derived datasets?

Processing At4g00755 antibody-derived datasets requires specific computational approaches tailored to plant research contexts:

ChIP-seq analysis pipeline:

• Quality control: FastQC for raw sequence data assessment

• Alignment: Bowtie2 or BWA against Arabidopsis thaliana reference genome

• Peak calling: MACS2 with FDR < 0.05 and fold enrichment > 2

• Normalization: Use spike-in controls to account for global epitope abundance changes

• Visualization: Create average occupancy profiles centered on transcription start sites

• Gene annotation: Associate peaks with genomic features using HOMER or BEDTools

Integrative analysis techniques:

• Differential binding analysis: Compare At4g00755 binding between conditions using DiffBind or MAnorm

• Motif analysis: Use MEME-ChIP or HOMER to identify enriched sequence motifs

• Functional enrichment: Identify biological processes enriched in target genes using GO analysis

• Network analysis: Construct gene regulatory networks using Cytoscape

• Co-occupancy analysis: Compare with publicly available ChIP-seq datasets for other factors

Recommended visualizations:

• Heat maps showing binding intensity across genes

• Scatter plots comparing antibody signals (e.g., H3-normalized H4K5ac vs. H3K9ac) as used in NuA4 studies

• Genome browser tracks showing binding patterns at specific loci

• Box plots comparing binding intensities across gene categories

These analyses help extract meaningful biological insights from complex ChIP-seq datasets generated using At4g00755 antibody.

How might At4g00755 antibody be used in conjunction with emerging techniques like proximity labeling?

Combining At4g00755 antibody with emerging proximity labeling techniques offers powerful new approaches for understanding protein interactions and functions:

BioID/TurboID applications:

• Create fusion proteins of At4g00755 with biotin ligases (BioID2 or TurboID)

• Express in Arabidopsis under native or inducible promoters

• Use At4g00755 antibody to validate expression levels and localization

• Perform proximity labeling followed by streptavidin pulldown

• Identify biotinylated proteins using mass spectrometry

APEX2 approaches:

• Generate At4g00755-APEX2 fusion constructs

• Validate expression using At4g00755 antibody

• Perform rapid proximity labeling with H₂O₂ and biotin-phenol

• Identify labeled proteins and compare with immunoprecipitation results

Split-BioID for interaction verification:

• Create split-BioID constructs with At4g00755 and potential interactors

• Validate using At4g00755 antibody

• Identify interaction-dependent biotinylation events

Methodological considerations:

• Use At4g00755 antibody to isolate native protein complexes for comparison

• Perform proximity labeling under different conditions (developmental stages, stress treatments)

• Validate key interactions using traditional co-IP with At4g00755 antibody

This integrated approach can reveal the dynamic interactome of At4g00755 under various conditions, providing insights into its functional roles in plant cells.

What potential exists for developing structure-based studies using At4g00755 antibody?

Structure-based studies utilizing At4g00755 antibody could provide valuable insights into F-box protein function in plants:

Cryo-EM structural analysis:

• Use At4g00755 antibody to purify native protein complexes

• Optimize sample preparation for cryo-EM analysis

• Determine structure of At4g00755 in complex with SCF components

• Compare with structures of other F-box protein complexes

X-ray crystallography approaches:

• Use At4g00755 antibody for immunoaffinity purification

• Generate Fab fragments from the antibody for co-crystallization

• Solve structure of At4g00755-Fab complex

• Identify key structural domains and interaction interfaces

Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

• Compare deuterium uptake in free vs. antibody-bound At4g00755

• Identify regions involved in conformational changes

• Map potential regulatory domains

Structure-guided functional studies:

• Based on structural insights, design targeted mutations in key domains

• Generate transgenic plants expressing mutant variants

• Use At4g00755 antibody to compare expression and binding properties

• Correlate structural features with functional outcomes

Drawing from approaches used in structural studies of protein complexes like G-protein signaling components and P-Rex1-Gβγ , these methods could reveal how At4g00755 recognizes its substrates and interacts with other SCF components in plant cells.

How can At4g00755 antibody contribute to understanding plant stress signaling networks?

At4g00755 antibody can serve as a powerful tool for elucidating the role of this F-box protein in plant stress signaling networks:

Stress-responsive phosphorylation studies:

• Perform immunoprecipitation with At4g00755 antibody under normal and stress conditions

• Analyze phosphorylation changes using phospho-specific antibodies or mass spectrometry

• Identify kinases responsible for stress-induced modifications

• Map phosphorylation sites to functional domains

Protein degradation dynamics:

• Track At4g00755 protein levels during stress responses using the antibody

• Identify stress conditions that trigger changes in protein abundance

• Correlate with ubiquitination status of potential target proteins

• Determine half-life changes under different stress conditions

Interactome shifts under stress:

• Compare At4g00755 immunoprecipitation results between normal and stress conditions

• Identify stress-specific interacting partners

• Map interactions to known stress signaling pathways

• Validate key interactions using reciprocal co-immunoprecipitation

Signal integration analysis:

• Use At4g00755 antibody-based ChIP-seq to map genomic binding sites

• Compare binding patterns under multiple stress conditions

• Identify common and stress-specific targets

• Construct network models of At4g00755-mediated stress responses

By implementing these approaches, researchers can determine how At4g00755 functions as a regulatory node in plant stress signaling networks, potentially identifying novel targets for improving crop stress resilience.

What are the implications of using At4g00755 antibody in studying circadian rhythm regulation in plants?

Using At4g00755 antibody to investigate potential connections to circadian rhythm regulation could reveal novel regulatory mechanisms:

Temporal expression profiling:

• Collect plant samples at regular intervals over 24-48 hours

• Use At4g00755 antibody to quantify protein abundance changes

• Compare with transcript oscillations to identify post-transcriptional regulation

• Correlate with known circadian phases

Protein-protein interaction dynamics:

• Perform immunoprecipitation with At4g00755 antibody at different circadian time points

• Identify time-of-day-specific interaction partners

• Focus on known circadian clock components (e.g., CCA1, LHY, TOC1)

• Validate interactions using reciprocal co-immunoprecipitation

Target protein degradation rhythms:

• Identify potential substrates of At4g00755-containing SCF complexes

• Track their abundance over circadian cycles using specific antibodies

• Correlate with At4g00755 expression patterns

• Perform degradation assays at different circadian times

Chromatin association dynamics:

• Perform ChIP-seq with At4g00755 antibody at different circadian times

• Identify rhythmic binding patterns

• Correlate with chromatin state changes

• Map to circadian-regulated genes

This circadian-focused approach could reveal whether At4g00755 functions in timing protein degradation events in the plant circadian system, potentially connecting ubiquitin-mediated proteolysis to temporal regulation of plant physiology and development.

What sample preparation techniques maximize protein recovery for At4g00755 antibody applications in challenging plant tissues?

Optimizing protein extraction from challenging plant tissues is critical for successful At4g00755 antibody applications:

Specialized extraction protocols for different tissues:

General optimization strategies:

• Buffer composition:

  • Test different pH ranges (7.0-8.0)

  • Optimize salt concentration (100-500mM NaCl)

  • Include detergents (0.1-1% Triton X-100 or NP-40)

• Physical disruption methods:

  • Compare mortar and pestle, bead beating, and sonication

  • Optimize tissue:buffer ratio (typically 1:3-1:5)

  • Test freeze-thaw cycles with liquid nitrogen

• Protein concentration techniques:

  • TCA/acetone precipitation

  • Methanol/chloroform precipitation

  • Ultrafiltration devices (various MWCO)

• Storage considerations:

  • Add glycerol (10-20%) for cryoprotection

  • Aliquot samples to avoid freeze-thaw cycles

  • Store at -80°C for long-term stability

These optimized protocols ensure maximum recovery of At4g00755 protein while minimizing interference from plant-specific compounds that could affect antibody binding.

What strategies can improve the specificity and sensitivity of At4g00755 antibody in immunohistochemistry applications?

Enhancing At4g00755 antibody performance in immunohistochemistry requires specific optimizations for plant tissues:

Fixation optimization:

• Compare different fixatives:

  • 4% paraformaldehyde (standard)

  • Ethanol-acetic acid (3:1)

  • Farmer's fixative

  • Carnoy's solution

• Test fixation times (2-24 hours)

• Optimize penetration with vacuum infiltration cycles

Antigen retrieval methods:

• Heat-induced epitope retrieval:

  • Citrate buffer (pH 6.0)

  • Tris-EDTA buffer (pH 9.0)

  • Microwave vs. pressure cooker methods

• Enzymatic retrieval:

  • Proteinase K (1-10 μg/ml)

  • Trypsin digestion

• Detergent permeabilization:

  • Triton X-100 (0.1-1%)

  • Saponin (0.01-0.1%)

Signal amplification techniques:

• Tyramide signal amplification

• Polymer-based detection systems

• Biotin-streptavidin amplification

Background reduction strategies:

• Pre-absorption of antibody with plant extract from At4g00755 knockout plants

• Increased blocking time (overnight at 4°C)

• Test different blocking agents:

  • BSA (1-5%)

  • Normal serum (5-10%)

  • Commercial blocking reagents

Controls and validation:

• At4g00755 knockout tissue as negative control

• At4g00755 overexpression tissue as positive control

• Peptide competition assay to confirm specificity

• Dual labeling with organelle markers to confirm subcellular localization

These optimizations will help researchers achieve specific and sensitive detection of At4g00755 protein in plant tissues while minimizing background and false positive signals.