At5g39460 Antibody

What is At5g39460 and why would a researcher need an antibody against it?

At5g39460 is a gene in Arabidopsis thaliana that encodes a putative F-box family protein according to the Arabidopsis Information Resource (TAIR) and UniProt databases . F-box proteins function as substrate recognition components within SKP1-CUL1-F-box (SCF) ubiquitin ligase complexes, which tag specific proteins for degradation via the ubiquitin-proteasome system.

Researchers would develop antibodies against At5g39460 to:

• Determine its spatial and temporal expression patterns

• Identify protein-protein interactions, particularly with SCF complex components

• Track protein turnover and stability

• Investigate its potential role in plant development or stress responses

• Confirm knockout or overexpression in transgenic lines

The development of specific antibodies against At5g39460 allows researchers to move beyond transcript-level analysis to directly study protein dynamics and interactions.

How should I design the immunogen for generating an At5g39460-specific antibody?

Designing an optimal immunogen is critical for antibody specificity, particularly for plant F-box proteins which often share conserved domains. Consider the following methodological approach:

Best practice recommendation:

• Perform sequence alignment with other Arabidopsis F-box proteins to identify unique regions

• Use epitope prediction algorithms to identify surface-exposed regions

• Avoid transmembrane domains or regions with post-translational modifications

• Consider coupling the chosen peptide/protein to a carrier protein like KLH or BSA

• For recombinant proteins, use bacterial expression systems with appropriate tags for purification

When designing peptide immunogens, aim for regions with minimal homology to other F-box family members to reduce cross-reactivity .

What are the key differences between polyclonal and monoclonal antibodies for plant protein research?

The choice between polyclonal and monoclonal antibodies affects experimental outcomes significantly in plant research:

Recent research indicates that recombinant antibodies outperform both traditional monoclonal and polyclonal antibodies in specificity tests with knockout controls. The YCharOS group demonstrated that recombinant antibodies showed 15-20% higher specificity across multiple applications compared to other antibody types .

For At5g39460 research, polyclonal antibodies provide a practical starting point, but recombinant antibodies offer superior reproducibility for long-term studies.

What are the optimal protein extraction methods when using antibodies against Arabidopsis F-box proteins like At5g39460?

F-box proteins present unique extraction challenges due to their involvement in protein-protein interactions and typically low abundance. The following protocol is optimized for At5g39460 detection:

Recommended Extraction Protocol:

• Harvest young Arabidopsis tissue (preferably 7-14 day seedlings) and flash-freeze in liquid nitrogen

• Grind tissue to fine powder using mortar and pestle kept at liquid nitrogen temperature

• Extract with buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 10% glycerol

  • 1 mM EDTA

  • 1 mM PMSF

  • Protease inhibitor cocktail

  • Critical component: 50 μM MG132 (proteasome inhibitor)

  • 10 mM N-ethylmaleimide (preserves ubiquitination)

• Centrifuge at 14,000 × g for 15 minutes at 4°C

• Transfer supernatant to fresh tube, add 5× SDS sample buffer

• Heat at 95°C for 5 minutes

Important considerations:

• F-box proteins typically have short half-lives; proteasome inhibitors significantly improve detection

• Extraction buffer should be optimized based on subcellular localization (nuclear, cytoplasmic, etc.)

• For difficult samples, TCA/acetone precipitation can remove interfering compounds common in plant tissues

• Protein extraction buffer AS08 300 has been successfully used for Arabidopsis protein extraction prior to western blotting

Recent studies have shown that including MG132 in extraction buffers improves F-box protein recovery by up to 3-fold, while the addition of N-ethylmaleimide helps preserve ubiquitination states that may be biologically significant .

How should I validate the specificity of an antibody against At5g39460?

Comprehensive validation is essential for antibody-based research reliability. For At5g39460, implement the "five pillars" validation approach:

1. Genetic strategies:

• Test on T-DNA insertion lines with disrupted At5g39460 (available from TAIR)

• Compare with CRISPR/Cas9-generated knockout lines

• Use overexpression lines as positive controls

• Expected outcome: Signal absent in knockout, enhanced in overexpression

2. Orthogonal strategies:

• Compare protein levels detected by antibody with At5g39460 transcript levels (qPCR)

• Correlate with RNA-seq data from public databases

• Expected outcome: General correlation between transcript and protein levels, with potential temporal offsets

3. Independent antibody strategies:

• Use multiple antibodies targeting different regions of At5g39460

• Compare antibodies from different sources or production methods

• Expected outcome: Similar detection patterns with some epitope-specific differences

4. Tagged protein expression:

• Generate transgenic lines expressing epitope-tagged At5g39460 (GFP, FLAG, etc.)

• Compare antibody signal with tag-specific antibody detection

• Expected outcome: Co-localization and similar expression patterns

5. Immunocapture mass spectrometry:

• Perform immunoprecipitation with the At5g39460 antibody

• Analyze precipitated proteins by mass spectrometry

• Expected outcome: Identification of At5g39460 and known interacting partners

The YCharOS study demonstrated that genetic knockout validation is the most definitive approach, with 95% specificity compared to 75% for other methods. This study revealed that approximately 12 publications per protein target included data from antibodies that failed to recognize the relevant target .

What controls are essential when performing western blots with At5g39460 antibodies?

Proper controls are crucial for interpreting western blot results with plant protein antibodies:

For F-box proteins specifically, the proteasome inhibition control is particularly informative. Young et al. (2019) demonstrated that proteasome inhibition with MG132 increased detection of several plant F-box proteins by 2-4 fold in Arabidopsis samples, confirming both antibody specificity and protein regulation by the ubiquitin-proteasome system .

How can I use At5g39460 antibodies to study protein-protein interactions within the SCF complex?

F-box proteins function through dynamic protein interactions that can be captured using antibody-based techniques:

Co-immunoprecipitation strategies for SCF complex analysis:

• Standard co-IP protocol:

  • Extract proteins under native conditions with 0.5% NP-40

  • Immunoprecipitate with anti-At5g39460 antibody

  • Probe western blots for SCF components (ASK1/SKP1, CUL1, RBX1)

  • Also probe for putative substrates under MG132 treatment

• Reciprocal co-IP validation:

  • Perform parallel IPs with antibodies against ASK1/SKP1

  • Probe for At5g39460 to confirm complex formation

  • Quantify interaction stoichiometry by densitometry

• Tandem affinity purification:

  • Generate transgenic plants expressing TAP-tagged At5g39460

  • Perform sequential purification steps

  • Identify partners by mass spectrometry

  • Validate key interactions with specific antibodies

Studies of other F-box proteins have shown that substrate identification often requires proteasome inhibition with MG132, as the interactions are typically transient. Additionally, crosslinking with 0.5-1% formaldehyde prior to extraction can stabilize weak interactions that might otherwise be lost during purification .

How can I resolve discrepancies between antibody detection and transcript levels of At5g39460?

Discrepancies between protein and transcript levels are common for F-box proteins and often reflect biological regulation rather than technical issues:

Systematic approach to resolve discrepancies:

• Confirm antibody specificity:

  • Verify using knockout controls

  • Test detection in multiple tissues and conditions

  • Ensure appropriate extraction conditions

• Investigate protein stability:

  • Perform cycloheximide chase experiments to measure protein half-life

  • Compare stability ± proteasome inhibitors (MG132)

  • Check for autoubiquitination (common in F-box proteins)

• Examine post-transcriptional regulation:

  • Check for known miRNA targeting sites in At5g39460 transcript

  • Assess transcript stability with actinomycin D treatment

  • Investigate alternative splicing variants

• Quantify translational efficiency:

  • Perform polysome profiling to assess translation rate

  • Compare against global translation patterns

  • Consider tissue-specific translational regulation

Recent research on plant F-box proteins has revealed that many undergo rapid turnover through autoubiquitination, resulting in protein half-lives of 1-4 hours despite stable mRNA levels. This regulation mechanism allows for rapid adjustment of F-box protein levels in response to environmental or developmental signals .

How can I perform immunolocalization of At5g39460 in plant tissues?

Immunolocalization of plant proteins requires specialized techniques to overcome cell wall barriers and preserve epitope accessibility:

Optimized immunolocalization protocol for At5g39460:

• Sample preparation:

  • Fix young Arabidopsis tissues in 4% paraformaldehyde in PBS (pH 7.4) for 2 hours at room temperature

  • Embed in paraffin or prepare for cryosectioning (10-15 μm sections)

  • Perform antigen retrieval with citrate buffer (pH 6.0) at 95°C for 10 minutes

  • Block with 5% BSA, 0.3% Triton X-100 in PBS for 1 hour

• Antibody incubation:

  • Primary: Incubate with anti-At5g39460 antibody (1:100-1:500 dilution) overnight at 4°C

  • Washing: PBS with 0.1% Tween-20, 3 × 10 minutes

  • Secondary: Fluorescently-labeled anti-rabbit IgG (1:500) for 2 hours at room temperature

  • Counterstain nuclei with DAPI (1 μg/ml)

• Controls and validation:

  • Negative control: Sections from At5g39460 knockout plants

  • Peptide competition: Pre-incubate antibody with immunizing peptide

  • Co-localization: Double-label with markers for relevant compartments (nuclear, cytoplasmic, etc.)

For plant proteins, immunolocalization results can be affected by fixation conditions. Comparative studies show that a combination of chemical fixation followed by enzyme-based cell wall digestion can improve antibody penetration while maintaining tissue morphology. Notably, technical validation with epitope-tagged proteins shows better concordance with immunofluorescence results in 70% of cases compared to transcriptional reporters .

Why am I detecting multiple bands with my At5g39460 antibody and how should I interpret them?

Multiple bands in immunoblots of plant F-box proteins can reflect biological complexity rather than non-specificity:

Common causes of multiple bands for F-box proteins:

• Post-translational modifications:

  • Phosphorylation: Often creates 2-8 kDa shifts

  • Ubiquitination: Ladder or smear of higher molecular weight bands

  • SUMOylation: Discrete bands ~15-17 kDa larger than main band

• Protein processing:

  • Alternative translation start sites

  • Proteolytic cleavage (partial degradation)

  • Alternative splicing variants

• Protein complexes:

  • Incompletely denatured protein complexes

  • Stable protein-protein interactions

Validation approaches to distinguish specific from non-specific bands:

An immunoprecipitation followed by mass spectrometry analysis could definitively identify the protein species present in each band. Studies with other F-box proteins have shown that the free form, SCF-bound form, and ubiquitinated forms can all be detected simultaneously in a single sample, resulting in a complex banding pattern .

How can I address weak or inconsistent signal when using At5g39460 antibodies?

Detecting plant F-box proteins can be challenging due to their typically low abundance and rapid turnover:

Systematic approach to improve signal:

• Protein extraction optimization:

  • Include proteasome inhibitors (50 μM MG132) during extraction

  • Use denaturing conditions to disrupt protein complexes

  • Add phosphatase inhibitors to preserve all protein forms

  • Include reducing agents to maintain epitope accessibility

• Sample enrichment strategies:

  • Concentrate proteins by TCA precipitation

  • Perform subcellular fractionation if localization is known

  • Use immunoprecipitation followed by western blot

  • Consider using plant tissues with higher expression (based on transcriptomics data)

• Signal enhancement methods:

  • Use high-sensitivity ECL substrates for western blots

  • Try fluorescent secondary antibodies with digital imaging

  • Increase antibody concentration (titrate from 1:500 to 1:100)

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

  • Consider signal amplification systems (tyramide, polymer-based)

• Technical considerations:

  • Ensure transfer efficiency (verify with reversible stain)

  • Optimize blocking conditions (BSA vs. milk, concentration)

  • Consider using PVDF rather than nitrocellulose membranes

  • Minimize washing stringency if signal is weak

Recent studies have shown that MG132 treatment can increase F-box protein detection by 3-5 fold compared to untreated samples, making it an essential component when working with these inherently unstable proteins .

How can I minimize cross-reactivity with other F-box proteins when using At5g39460 antibodies?

Cross-reactivity is a significant concern when working with members of large protein families like F-box proteins (Arabidopsis contains >700 F-box genes):

Strategies to minimize and assess cross-reactivity:

• Antibody design and selection:

  • Target unique regions outside the conserved F-box domain

  • Use peptide arrays to test cross-reactivity against similar proteins

  • Consider multiple antibodies targeting different epitopes

  • Pre-absorb antibody with recombinant proteins from close homologs

• Experimental validation:

  • Test antibody on overexpression lines of related F-box proteins

  • Perform immunoprecipitation followed by mass spectrometry

  • Compare reactivity across multiple tissues with known expression profiles

  • Use epitope-tagged versions as positive controls

• Data interpretation safeguards:

  • Always include knockout/knockdown controls

  • Compare results with transcriptomic data

  • Consider using differential expression across tissues/conditions

  • Be cautious with absolute quantification

A systematic analysis by the YCharOS study demonstrated that approximately 50-75% of antibodies show some degree of cross-reactivity within protein families, emphasizing the importance of comprehensive validation. Their data showed that KO cell lines provide the most definitive validation, particularly for immunofluorescence applications where cross-reactivity was observed in up to 40% of tested antibodies .

How can new antibody technologies advance At5g39460 research beyond traditional applications?

Emerging technologies offer new possibilities for studying plant F-box proteins with increased precision:

Advanced antibody technologies applicable to At5g39460 research:

• Single-domain antibodies (nanobodies):

  • Smaller size allows better tissue penetration in plants

  • Can be expressed in vivo as "intrabodies"

  • Enable super-resolution microscopy of native proteins

  • Can be used to track protein dynamics in living tissues

• Proximity-dependent labeling:

  • Antibody-enzyme conjugates (APEX, BioID, TurboID)

  • Map protein neighborhoods in native context

  • Identify transient interactions with substrates

  • Spatial mapping of protein interactions in specific cell types

• Antibody-based protein modulation:

  • Targeted protein degradation systems

  • Conformation-specific antibodies to trap functional states

  • Optogenetic control of protein function via antibody-based tethering

  • Targeted interactome rewiring

• Single-cell applications:

  • Immuno-based single-cell sorting

  • Spatial transcriptomics with protein correlation

  • Multi-parameter single-cell analysis

Recent developments in AI-guided antibody design have significantly improved target specificity. The MAGE (Monoclonal Antibody GEnerator) system demonstrated the capacity to generate paired antibody sequences with experimentally validated binding specificity, which could be adapted for plant protein targets like At5g39460 .

How can antibody-based technologies be combined with genomic approaches to study At5g39460 function?

Integrating antibody methods with genomic technologies provides comprehensive functional insights:

Integrated research approaches:

• ChIP-seq applications:

  • If At5g39460 influences transcription, ChIP-seq can map genomic targets

  • Requires high-quality antibodies or epitope-tagged constructs

  • Can reveal condition-specific binding patterns

  • Integration with RNA-seq data validates functional impacts

• Proteogenomic strategies:

  • Correlate protein levels (antibody-based) with transcript profiles

  • Map post-translational modifications using specific antibodies

  • Create protein-centric regulatory networks

  • Identify discordance between transcript and protein as regulatory targets

• Antibody-guided CRISPR screens:

  • Use antibody-based phenotypic readouts for genetic screens

  • Identify genes affecting At5g39460 stability or localization

  • Screen for modifiers of At5g39460-dependent processes

  • Validate hits with reverse genetic approaches

• Multi-omics data integration:

  • Antibody-validated protein interactions

  • Transcriptional responses to protein perturbation

  • Metabolic changes associated with protein function

  • Mathematical modeling of integrated datasets

Studies combining antibody-based protein quantification with transcriptomics have revealed that protein abundance explains approximately 70% of phenotypic variation in plants, compared to only 40% for transcript levels alone, highlighting the importance of direct protein measurements .

How might antibody research on At5g39460 contribute to understanding broader F-box protein functions in plants?

Research on At5g39460 can serve as a model for investigating the entire F-box protein family:

Comparative approaches with broader impact:

• Family-wide epitope mapping:

  • Systematic analysis of antibody cross-reactivity

  • Identification of conserved and variable epitopes

  • Development of subfamily-specific antibodies

  • Creation of antibody toolkits for F-box protein research

• Structural and functional conservation:

  • Compare interaction patterns across F-box protein subfamilies

  • Identify conserved post-translational modifications

  • Map substrate recognition domains using antibody blocking

  • Trace evolutionary relationships through antibody cross-reactivity

• Systems-level understanding:

  • Map F-box protein networks using antibody-based methods

  • Quantify dynamics of multiple F-box proteins simultaneously

  • Identify functional redundancy through parallel tracking

  • Develop predictive models of F-box protein function

• Translational applications:

  • Transfer knowledge to crops and other plant species

  • Develop antibody tools for agricultural applications

  • Create diagnostic tools for plant developmental states

  • Enable precise phenotyping of plant responses

A comprehensive antibody analysis demonstrated that approximately 31.11% of patients with systemic sclerosis exhibited seropositivity for specific antibodies, suggesting that even low-abundance proteins can serve as critical biomarkers when appropriate antibody tools are developed . Similar approaches could identify plant F-box proteins serving as markers for specific developmental or stress responses.

What are the most reliable resources for validating At5g39460 antibodies?

Several databases and resources can assist in antibody validation for plant proteins: