At1g10780 Antibody

What is the At1g10780 protein and why is it significant in plant research?

At1g10780 encodes an F-box/RNI-like superfamily protein in Arabidopsis thaliana . F-box proteins are components of SCF ubiquitin-ligase complexes that regulate various cellular processes through targeted protein degradation. This particular F-box protein has orthologs in multiple plant species including Solanum lycopersicum and Momordica charantia, suggesting evolutionary conservation . Understanding its function can provide insights into protein degradation pathways and their roles in plant development, stress responses, and signaling networks.

How specific is the At1g10780 antibody for its target protein?

The At1g10780 antibody (CSB-PA890292XA01DOA) is generated against the Q9SAC4 protein in Arabidopsis thaliana . Cross-reactivity testing data indicates high specificity for the target protein in Arabidopsis samples, though researchers should perform validation in their specific experimental systems. When comparing to other commercial plant antibodies, which typically show >80% specificity when properly validated, the At1g10780 antibody performs similarly. Cross-reactivity with homologous proteins in closely related plant species may occur due to sequence conservation and should be experimentally determined before using the antibody in non-Arabidopsis systems.

What validation methods should be used to confirm At1g10780 antibody specificity?

For rigorous validation, researchers should implement multiple approaches:

• Western blot analysis with appropriate controls:

  • Wild-type Arabidopsis tissue expressing At1g10780

  • Knockout/knockdown lines (at1g10780 mutants)

  • Overexpression lines

  • Recombinant protein as positive control

• Immunoprecipitation followed by mass spectrometry

• Immunolocalization in wild-type versus mutant tissues

• Preabsorption test with the immunizing peptide/protein

What are the optimal protocols for using At1g10780 antibody in Western blot applications?

For optimal Western blot results when using the At1g10780 antibody:

• Sample preparation:

  • Extract total protein from Arabidopsis tissues using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail

  • Use 20-50 μg of total protein per lane

• Electrophoresis and transfer:

  • Separate proteins on 10-12% SDS-PAGE

  • Transfer to PVDF membrane (nitrocellulose is less effective for this protein)

• Immunoblotting:

  • Block with 5% non-fat dry milk in TBS-T for 1 hour at room temperature

  • Incubate with At1g10780 antibody at 1:1000 dilution overnight at 4°C

  • Wash 4 times with TBS-T, 5 minutes each

  • Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

  • Develop using ECL reagent

This protocol has been optimized based on approaches similar to those used for other plant F-box proteins and the PsaF antibody methodology described in reference .

Can At1g10780 antibody be used effectively for chromatin immunoprecipitation (ChIP) experiments?

While the At1g10780 antibody has not been specifically validated for ChIP, similar approaches to those used for other plant proteins can be adapted. Based on successful ChIP protocols used for LEC1 and BDR proteins in Arabidopsis :

• Crosslinking and chromatin preparation:

  • Crosslink plant tissue with 1% formaldehyde for 10 minutes under vacuum

  • Quench with 125 mM glycine

  • Extract and sonicate chromatin to 200-500 bp fragments

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate with At1g10780 antibody (5-10 μg) overnight at 4°C

  • Capture with protein A/G beads

  • Wash with increasing stringency buffers

  • Reverse crosslinks and purify DNA

• Controls and validation:

  • Include IgG negative control

  • Use target gene knockout as biological negative control

  • Validate enrichment by qPCR of known targets before sequencing

Since F-box proteins primarily function in protein degradation rather than direct DNA binding, researchers should consider whether At1g10780 actually associates with chromatin. If pursuing ChIP experiments, validation experiments are crucial before proceeding to genome-wide analyses.

What are the recommended protocols for immunolocalization of At1g10780 in plant tissues?

For effective immunolocalization of At1g10780:

• Tissue preparation:

  • Fix tissue in 4% paraformaldehyde in PBS for 1 hour under vacuum

  • Embed in paraffin or prepare for cryosectioning

  • Section to 8-10 μm thickness

• Immunostaining:

  • Deparaffinize or rehydrate sections

  • Perform antigen retrieval (10 mM sodium citrate, pH 6.0, 95°C for 10 minutes)

  • Block with 3% BSA, 0.3% Triton X-100 in PBS for 1 hour

  • Incubate with At1g10780 antibody (1:100-1:200) overnight at 4°C

  • Wash 3 times with PBS

  • Apply fluorescent secondary antibody (1:200-1:500) for 1-2 hours at room temperature

  • Counterstain with DAPI for nuclei visualization

  • Mount with anti-fade mounting medium

• Controls:

  • Include secondary-only control

  • Use knockout/knockdown plants as negative control

  • Consider co-localization with known compartment markers such as ARF1 for Golgi

This protocol draws on approaches used for immunolocalization of other plant proteins, with modifications specific to F-box protein detection.

How should researchers interpret Western blot results when multiple bands appear with the At1g10780 antibody?

Multiple bands in Western blots can result from various biological and technical factors:

For proper interpretation:

• Compare with knockout controls to identify specific bands

• Consider alternative splicing - analyze RNA-seq data for evidence of splice variants

• Test different extraction methods to minimize degradation

• If post-translational modifications are suspected, use specific inhibitors to confirm

Since F-box proteins often undergo dynamic regulation and can be involved in complex formation, careful validation is essential to distinguish biologically relevant signals from technical artifacts.

What factors most commonly affect At1g10780 antibody performance in immunoprecipitation experiments?

Several factors can significantly impact immunoprecipitation efficiency:

• Buffer composition:

  • Ionic strength affects antibody-antigen interaction

  • Detergent type and concentration must balance solubilization and epitope preservation

  • Recommended starting buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA with protease inhibitors

• Antibody amount and quality:

  • Optimal antibody:protein ratio must be determined empirically

  • Typically start with 2-5 μg antibody per 500 μg total protein

  • Antibody storage conditions affect performance

• Crosslinking considerations:

  • For transient interactions, consider chemical crosslinking with DSP or formaldehyde

  • Crosslinking may mask epitopes - test different conditions

• Bead selection:

  • Protein A vs. Protein G affinity varies with antibody isotype

  • Pre-clearing steps reduce non-specific binding

For optimal At1g10780 immunoprecipitation, researchers should systematically optimize these parameters using positive and negative controls to assess specificity and efficiency.

How can researchers verify that the observed protein patterns in their experiments truly represent At1g10780 function?

To confirm that observed patterns truly reflect At1g10780 function:

• Genetic approaches:

  • Compare at1g10780 knockout/knockdown lines with wild-type plants

  • Complement mutant phenotypes with the wild-type gene

  • Use multiple independent mutant alleles to rule out background effects

  • Create rescue lines with tagged versions for antibody validation

• Biochemical validation:

  • Perform in vitro ubiquitination assays to confirm F-box protein activity

  • Identify interaction partners through mass spectrometry

  • Verify protein stability changes in mutant vs. wild-type plants

• Transcriptomic analysis:

  • Analyze expression changes in mutant lines

  • Compare with published datasets for related F-box proteins

  • Look for enrichment of specific pathways

• Additional antibody-based validations:

  • Use multiple antibodies targeting different epitopes if available

  • Perform siRNA/CRISPR knockdown followed by antibody testing

This multi-faceted approach provides robust validation of experimental observations and helps distinguish direct effects of At1g10780 from indirect or non-specific effects.

How can the At1g10780 antibody be used to investigate protein-protein interactions in the ubiquitin-proteasome pathway?

The At1g10780 F-box protein likely functions within SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes. To investigate these interactions:

• Co-immunoprecipitation strategies:

  • Use At1g10780 antibody to pull down the protein complex

  • Analyze co-precipitated proteins by mass spectrometry

  • Confirm interactions with reciprocal co-IPs using antibodies against putative partners

  • Consider gentle extraction conditions to preserve weak interactions

• Proximity-based labeling approaches:

  • Create transgenic plants expressing At1g10780 fused to BioID or TurboID

  • Identify proteins that become biotinylated when in proximity

  • Compare with immunoprecipitation results for validation

• Yeast two-hybrid screening validation:

  • Use At1g10780 antibody to confirm expression of identified interactors in planta

  • Verify subcellular co-localization with interaction partners

• In vivo dynamics:

  • Monitor At1g10780 levels under different conditions

  • Track substrates using cycloheximide chase assays combined with At1g10780 antibody detection

This comprehensive approach builds on principles of protein interaction studies while leveraging the specificity of the At1g10780 antibody to validate and extend findings in physiologically relevant contexts.

How can researchers effectively use the At1g10780 antibody in comparative studies across different plant species?

For cross-species applications:

• Sequence homology assessment:

  • Analyze epitope conservation across species

  • Perform sequence alignment of At1g10780 orthologs

  • Create a phylogenetic tree to understand evolutionary relationships

• Cross-reactivity testing:

  • Perform Western blots with protein extracts from multiple species

  • Include positive (Arabidopsis) and negative controls

  • Consider dot blots with recombinant proteins from different species

• Optimization strategies for non-Arabidopsis species:

  • Adjust extraction buffers based on tissue composition

  • Modify antibody concentration and incubation conditions

  • Test different blocking agents to reduce background

• Data interpretation across species:

  • Account for differences in protein size due to sequence variations

  • Consider differences in post-translational modifications

  • Analyze expression patterns in the context of species-specific physiology

Based on homologs identified in Solanum lycopersicum and Momordica charantia , the At1g10780 antibody might be applicable for comparative studies in these species after proper validation.

What approaches can be used to study At1g10780 protein dynamics during plant development and stress responses?

To investigate At1g10780 dynamics:

• Developmental profiling:

  • Collect tissues at defined developmental stages

  • Quantify At1g10780 protein levels by Western blot

  • Normalize to appropriate loading controls

  • Create protein expression maps across developmental stages

• Stress response analysis:

  • Subject plants to various stresses (drought, salt, pathogen, temperature)

  • Monitor At1g10780 protein levels at defined time points

  • Compare with transcriptional changes using RT-qPCR

  • Analyze post-translational modifications induced by stress

• Subcellular localization changes:

  • Perform immunolocalization under different conditions

  • Track potential nuclear-cytoplasmic shuttling

  • Use subcellular fractionation followed by Western blot

• Protein stability assessment:

  • Measure protein half-life using cycloheximide chase assays

  • Compare stability under different conditions

  • Investigate proteasome-dependent degradation with inhibitors like MG132

This systematic approach provides comprehensive insights into how At1g10780 function is regulated in response to developmental and environmental signals.

How can researchers integrate At1g10780 antibody data with transcriptomic and functional genomics approaches?

For integrative studies combining antibody-based detection with other -omics approaches:

• Correlation of protein and transcript levels:

  • Compare Western blot quantification with RNA-seq or microarray data

  • Identify conditions where post-transcriptional regulation occurs

  • Calculate protein-mRNA correlation coefficients across conditions

• Integration with ChIP-seq data:

  • If At1g10780 affects transcription factors (direct or indirect)

  • Compare binding profiles of transcription factors in wild-type vs. at1g10780 mutants

  • Analyze chromatin accessibility changes using ATAC-seq

• Network analysis:

  • Place At1g10780 in functional modules based on protein interaction data

  • Correlate with co-expression networks derived from transcriptomics

  • Identify regulatory hubs affected by At1g10780 function

• Phenomic integration:

  • Connect At1g10780 protein levels to phenotypic data using supervised machine learning

  • Build predictive models of plant phenotypes based on protein abundance

  • Validate with targeted genetic manipulations

This integrative approach, drawing on concepts from systems biology as described in reference , provides a comprehensive understanding of At1g10780 function within the broader context of plant biology.

What emerging technologies might enhance the application of At1g10780 antibody in plant research?

Several cutting-edge approaches hold promise for expanding At1g10780 antibody applications:

• Single-cell proteomics:

  • Adapting At1g10780 antibody for use in mass cytometry (CyTOF)

  • Integration with single-cell transcriptomics

  • Spatial proteomics in tissue sections

• Advanced microscopy techniques:

  • Super-resolution microscopy for precise localization

  • Live-cell imaging with nanobody derivatives

  • Correlative light and electron microscopy

• Proteoform-specific detection:

  • Development of antibodies specific to post-translationally modified variants

  • Multiplexed detection of different At1g10780 states

• Synthetic biology approaches:

  • Engineering sensor systems based on At1g10780 antibody fragments

  • Creating optogenetic tools to manipulate At1g10780 function

These emerging technologies will expand our understanding of At1g10780 function at unprecedented resolution and in novel contexts.

What considerations should guide experimental design when studying At1g10780 in different plant tissues and developmental stages?

When designing experiments across tissues and developmental stages:

• Sampling considerations:

  • Standardize harvesting times to control for circadian effects

  • Consider tissue-specific extraction protocols

  • Account for developmental heterogeneity within samples

• Normalization strategies:

  • Select appropriate reference proteins for each tissue/stage

  • Consider absolute quantification with recombinant protein standards

  • Implement statistical approaches to handle tissue-specific variability

• Experimental controls:

  • Include developmental series from multiple independent plants

  • Consider genetic background effects when using mutants

  • Account for environmental variables

• Data integration framework:

  • Develop consistent data collection and analysis pipelines

  • Establish clear metadata standards

  • Create visualization tools for complex developmental datasets