At1g43140 Antibody

What is At1g43140 and why is it important in Arabidopsis research?

At1g43140 is an Arabidopsis thaliana protein that functions as one of the closest orthologs to AtCUL1 (At4g02570), alongside four other proteins (At1g02980, At1g59800, and At1g59790) . These proteins belong to the cullin protein family, which plays critical roles in protein degradation through the ubiquitin-proteasome pathway. Understanding At1g43140 is important because cullin proteins are fundamental components of SCF (Skp1-Cullin-F-box) complexes that regulate numerous developmental processes in plants including embryogenesis, hormone signaling, and stress responses. Antibodies targeting At1g43140 allow researchers to study its expression patterns, subcellular localization, and potential functional redundancy with other cullin family members in Arabidopsis.

How is At1g43140 structurally and functionally related to AtCUL1?

At1g43140 shares significant structural homology with AtCUL1, particularly in the cullin homology domain that mediates interactions with other SCF complex components. While AtCUL1 is well-characterized and known to be essential for early embryogenesis in Arabidopsis , At1g43140 represents one of its potential functional homologs. The protein contains conserved domains necessary for RUB1/NEDD8 conjugation, similar to what has been observed with AtCUL1, which exists in both unconjugated and RUB1-conjugated isoforms . The structural similarity suggests At1g43140 may participate in protein degradation pathways, though potentially in different developmental contexts or cell types than AtCUL1. Antibodies targeting specific epitopes of At1g43140 are essential for distinguishing its expression and localization from other cullin family members.

What detection methods are most effective for At1g43140 antibodies in plant tissues?

For effective detection of At1g43140 in plant tissues, immunolocalization combined with confocal microscopy provides the most reliable spatial distribution data. Based on approaches used for related cullin proteins, a multi-method strategy is recommended:

• Western blotting with SDS-PAGE separation can reveal protein expression levels and post-translational modifications (such as RUB1 conjugation patterns) .

• Immunofluorescence microscopy enables subcellular localization studies, particularly important since cullin family proteins like AtCUL1 show both nuclear and cytoplasmic distribution .

• Chromatin immunoprecipitation (ChIP) may be useful if At1g43140 associates with chromatin-bound complexes.

• Immunoprecipitation followed by mass spectrometry can identify interaction partners unique to At1g43140 versus other cullin family members.

When designing these experiments, researchers should include appropriate controls with preimmune serum to distinguish specific from non-specific signals, as demonstrated in AtCUL1 localization studies .

How can researchers ensure antibody specificity when studying At1g43140 among closely related cullin family proteins?

Ensuring antibody specificity for At1g43140 amid closely related cullin family proteins requires a multi-faceted validation approach:

• Epitope selection strategy: Target unique regions of At1g43140 that differ from AtCUL1 and other orthologs. Computational analysis of protein sequences can identify divergent regions suitable for antibody generation. Recent advances in computational antibody design allow for in silico identification of potential epitopes with minimal cross-reactivity .

• Cross-reactivity testing: Validate antibody specificity using protein extracts from knockout/knockdown lines of At1g43140 and other cullin family members. Western blot analysis should demonstrate specific recognition of At1g43140 without detecting AtCUL1 or other orthologs.

• Recombinant protein controls: Express and purify recombinant versions of At1g43140 and related proteins to create a specificity panel for antibody validation. This approach has been successfully applied for antibody validation in plant systems, similar to the methods used for AtCUL1 antibody validation against tobacco cullins .

• Peptide competition assays: Pre-incubate antibodies with synthetic peptides representing the target epitope to confirm binding specificity. Signal diminution in the presence of the specific peptide confirms epitope specificity.

• Heterologous expression systems: Test antibody specificity in transgenic tobacco BY2 cells expressing tagged versions of At1g43140, similar to the approach used for AtCUL1 .

This comprehensive validation enables confident distinction between At1g43140 and its close relatives, even when studying tissues where multiple cullin family members are co-expressed.

What is the optimal immunization strategy for generating high-affinity At1g43140 antibodies?

Generating high-affinity antibodies against At1g43140 requires careful consideration of antigen preparation and immunization protocols:

• Antigen design options:

  • Full-length recombinant At1g43140 may present challenges due to its size and potential conformational complexity

  • Synthetic peptides (15-25 amino acids) from unique regions show higher success rates

  • Domain-specific recombinant fragments focusing on regions that differ from other cullin proteins

• Expression system selection: For full-length or domain proteins, E. coli systems with solubility tags (MBP, SUMO) improve antigen quality. For complex antigens, eukaryotic expression systems may better preserve native conformation.

• Immunization protocol: A proven approach involves:

  • Primary immunization with complete Freund's adjuvant

  • 3-4 boosters at 2-3 week intervals with incomplete Freund's adjuvant

  • Final boost without adjuvant 3-4 days before hybridoma generation

• Antibody class consideration: While IgG antibodies are typical for research applications, IgM monoclonals (similar to those developed for plant cell wall components like in the CCRC M36 antibody ) offer advantages when targeting carbohydrate-modified regions of At1g43140.

• Screening methodology: Initial ELISA screening followed by application-specific validation (Western blot, immunoprecipitation, immunofluorescence) ensures selection of clones suitable for intended research applications.

This strategy maximizes the likelihood of generating antibodies with sufficient specificity and affinity for reliable At1g43140 detection across multiple experimental platforms.

How can computational approaches improve At1g43140 antibody development and validation?

Computational methods offer significant advantages for both designing and validating At1g43140 antibodies:

• Structure-based epitope prediction: Using homology modeling based on known cullin structures to predict surface-exposed regions unique to At1g43140 enhances epitope selection. This approach decreases reliance on experimental trial and error for finding initial hits .

• Paratope optimization: Computational redesign of complementarity-determining regions (CDRs) can enhance antibody affinity and specificity. Recent advances in CDR redesign utilize highly developable antibody frameworks and modify original CDRs to recognize specific epitopes with improved affinity .

• Molecular dynamics simulations: Predicting antibody-antigen interaction stability through in silico methods helps prioritize candidates before experimental validation. This can significantly reduce development timelines and costs .

• Cross-reactivity prediction: Computational scanning of the Arabidopsis proteome can identify potential cross-reactive proteins based on epitope similarity, guiding experimental validation efforts.

• Developability assessment: In silico prediction of antibody biophysical properties (stability, solubility) can identify potential manufacturing or storage issues early in development. This represents a well-developed application of computational protein design in biotherapeutic discovery that can be adapted to research antibodies .

Implementation of these computational approaches can significantly accelerate At1g43140 antibody development while enhancing specificity and reducing reliance on extensive experimental screening.

What are the optimal fixation and permeabilization methods for At1g43140 immunolocalization in plant tissues?

Successful immunolocalization of At1g43140 in plant tissues requires careful optimization of fixation and permeabilization protocols. Based on methods established for AtCUL1 and other nuclear-cytoplasmic proteins in Arabidopsis:

• Fixation options:

  • Paraformaldehyde (4%) in PBS for 20-30 minutes preserves protein antigenicity while maintaining cellular structure

  • Methanol-acetone fixation (10 minutes at -20°C) may improve antibody accessibility to certain epitopes

  • Glutaraldehyde should be avoided or limited to 0.1-0.25% as higher concentrations may mask At1g43140 epitopes

• Permeabilization strategy:

  • For paraformaldehyde-fixed samples: 0.1-0.5% Triton X-100 in PBS (15-30 minutes)

  • For cell wall digestion: Pectolyase (1%) and cellulase (2%) treatment improves antibody penetration in intact tissues

  • Vacuum infiltration during both fixation and permeabilization enhances reagent penetration in leaf tissues

• Blocking conditions:

  • BSA (3-5%) with 0.1% Tween-20 in PBS minimizes background

  • Normal serum (5-10%) from the same species as secondary antibody

  • Extended blocking (2+ hours) reduces non-specific binding

• Antibody incubation parameters:

  • Primary antibody dilutions: Initial testing at 1:100, 1:500, and 1:1000

  • Overnight incubation at 4°C improves signal quality

  • Thorough washing (5× 10 minutes) between antibody steps is critical

These recommendations are based on successful immunolocalization protocols used for AtCUL1, which demonstrated both nuclear and cytoplasmic localization patterns in plant cells .

How can researchers distinguish between At1g43140 and its post-translationally modified forms?

Distinguishing between native At1g43140 and its post-translationally modified forms requires specialized experimental approaches:

• Detection of RUB1/NEDD8 conjugation:

  • High-resolution SDS-PAGE (8% gels) can separate unconjugated and RUB1-conjugated forms, as demonstrated with AtCUL1

  • Western blotting with both anti-At1g43140 and anti-RUB1 antibodies on parallel blots

  • Immunoprecipitation with anti-At1g43140 followed by anti-RUB1 Western blotting

• Phosphorylation analysis:

  • Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • Phosphatase treatment of samples prior to Western blotting to confirm phosphorylation

  • Phospho-specific antibodies targeting predicted phosphorylation sites

• Ubiquitination assessment:

  • Size-shift detection using anti-ubiquitin antibodies

  • Tandem ubiquitin binding entity (TUBE) pulldown followed by At1g43140 detection

  • Mass spectrometry to identify ubiquitination sites

• Tissue-specific modification patterns:

  • Compare modification profiles across different tissues and developmental stages

  • Analyze changes in modification under various stress conditions or hormone treatments

This approach enables comprehensive characterization of At1g43140 post-translational modifications that may regulate its function in different cellular contexts.

What are effective strategies for co-immunoprecipitation of At1g43140 protein complexes?

Effective co-immunoprecipitation (co-IP) of At1g43140 protein complexes requires preserving native protein interactions while achieving sufficient extraction and purification:

• Extraction buffer optimization:

  • Low-stringency buffers containing 0.5% NP-40 or 0.1% Triton X-100

  • Physiological salt concentrations (100-150 mM NaCl)

  • Protease inhibitor cocktail and phosphatase inhibitors

  • N-ethylmaleimide (NEM) to preserve ubiquitination

  • MG132 pretreatment of tissues to stabilize SCF complexes

• Antibody coupling strategies:

  • Direct coupling to Protein A/G magnetic beads for clean elution

  • Crosslinking antibodies to beads (using BS3 or DMP) to prevent antibody contamination

  • Pre-clearing lysates with beads alone to reduce non-specific binding

• Specialized approaches for transient interactions:

  • In vivo crosslinking with formaldehyde (1%, 10 minutes) prior to extraction

  • Proximity-dependent biotinylation (BioID) with At1g43140-BirA* fusion proteins

  • Two-step immunoprecipitation for increased specificity

• Validation methods:

  • Reciprocal co-IP with antibodies against known or suspected interaction partners

  • Competitive elution with antigenic peptides to confirm specificity

  • Mass spectrometry analysis of co-precipitated proteins

This comprehensive approach enables researchers to capture At1g43140 protein complexes while maintaining physiologically relevant interactions for functional characterization.

How should researchers quantify At1g43140 protein levels across different experimental conditions?

Accurate quantification of At1g43140 protein levels requires careful experimental design and appropriate normalization strategies:

• Western blot quantification:

  • Use gradient loading of known protein standards to create calibration curves

  • Apply fluorescent secondary antibodies for wider linear detection range

  • Employ image analysis software (ImageJ, Li-COR Image Studio) for densitometry

  • Normalize to stable reference proteins (NOT actin or tubulin, which may be SCF substrates)

  • Include technical triplicates and biological replicates (minimum n=3)

• ELISA-based quantification:

  • Develop sandwich ELISA using two antibodies recognizing different At1g43140 epitopes

  • Include recombinant At1g43140 standards for absolute quantification

  • Optimize sample dilutions to ensure measurements within the linear range

  • Perform spike recovery tests to validate quantification in complex matrices

• Mass spectrometry approaches:

  • Selected reaction monitoring (SRM) for targeted At1g43140 quantification

  • Use isotope-labeled peptide standards for absolute quantification

  • Monitor multiple peptides per protein for increased confidence

  • Normalize to invariant "housekeeping" proteins

• Statistical analysis requirements:

  • Apply appropriate statistical tests based on data distribution (parametric vs. non-parametric)

  • Correct for multiple comparisons when analyzing multiple conditions

  • Report effect sizes alongside p-values

  • Use mixed-effects models when analyzing time-course experiments

This methodical approach ensures reliable quantification of At1g43140 protein levels, enabling meaningful comparisons across different experimental conditions and treatments.

What control experiments are essential when using At1g43140 antibodies in chromatin immunoprecipitation (ChIP) studies?

Chromatin immunoprecipitation with At1g43140 antibodies requires rigorous controls to ensure valid interpretations:

• Antibody-specific controls:

  • IgG control from the same species as the At1g43140 antibody

  • Pre-immune serum control when available

  • Peptide competition assay to confirm epitope specificity

  • ChIP in At1g43140 knockout/knockdown lines as negative controls

• Technical validation controls:

  • Input chromatin samples (typically 1-5% of ChIP material)

  • Serial dilution of ChIP and input samples to confirm linear amplification

  • Sonication efficiency assessment via gel electrophoresis

  • Positive control regions (promoters of known SCF-regulated genes)

  • Negative control regions (transcriptionally inactive heterochromatin)

• Biological controls:

  • Parallel ChIP with antibodies against known SCF complex components

  • Treatment conditions known to affect cullin activity (proteasome inhibitors)

  • Developmental stages with differential At1g43140 expression

  • Tissue specificity controls when examining specific cell types

• Data analysis considerations:

  • Normalize ChIP-qPCR data to input samples

  • For ChIP-seq, include spike-in controls for normalization

  • Use appropriate peak-calling algorithms with FDR correction

  • Validate novel binding sites with orthogonal methods

Following these control practices ensures that ChIP data with At1g43140 antibodies accurately represents the genuine chromatin interactions of this protein rather than experimental artifacts or non-specific binding.

How can researchers differentiate between the functions of At1g43140 and other cullin family members through antibody-based approaches?

Differentiating between the functions of At1g43140 and other cullin family members requires strategic experimental design leveraging antibody specificity:

• Comparative immunoprecipitation strategy:

  • Perform parallel IPs with antibodies specific to each cullin family member

  • Analyze co-precipitated proteins by mass spectrometry to identify:

    • Shared binding partners (core SCF components)

    • Unique binding partners (specific F-box proteins, substrates)

  • Compare ubiquitination activity of immunoprecipitated complexes using in vitro assays

• Spatiotemporal expression mapping:

  • Conduct co-immunofluorescence studies with antibodies against multiple cullins

  • Analyze expression patterns across different tissues, cell types, and developmental stages

  • Quantify co-localization coefficients to determine degree of functional overlap

  • Examine subcellular localization differences, focusing on nuclear-cytoplasmic distribution patterns

• Perturbation-response experiments:

  • Monitor changes in At1g43140 vs. other cullins in response to:

    • Plant hormones (auxin, gibberellin, jasmonate)

    • Abiotic stresses (drought, salt, temperature)

    • Cell cycle progression

  • Compare post-translational modification patterns (especially RUB1/NEDD8 conjugation)

• Genetic complementation analysis:

  • Express epitope-tagged At1g43140 in AtCUL1 mutant backgrounds

  • Use antibodies against the epitope tag to monitor expression and localization

  • Assess rescue of phenotypic defects to determine functional redundancy

This systematic comparison enables attribution of specific functions to At1g43140 versus other cullin family members, elucidating their respective roles in plant development and stress responses.

How can advanced imaging techniques enhance At1g43140 antibody-based research?

Advanced imaging techniques offer new possibilities for studying At1g43140 localization, dynamics, and interactions:

• Super-resolution microscopy applications:

  • Structured illumination microscopy (SIM) provides 2x resolution improvement for detailed localization

  • Stochastic optical reconstruction microscopy (STORM) enables single-molecule localization precision

  • Stimulated emission depletion (STED) microscopy for enhanced visualization of At1g43140 in multiprotein complexes

  • These techniques can reveal previously undetectable subcellular patterns, similar to the detailed localization observed with AtCUL1

• Live-cell imaging approaches:

  • Combining anti-At1g43140 antibody fragments with cell-penetrating peptides

  • Using split fluorescent protein complementation to visualize interactions in vivo

  • Implementing optogenetic tools to manipulate At1g43140 function with light

  • These methods allow temporal analysis of At1g43140 dynamics during cell cycle progression and stress responses

• FRET/FLIM analysis:

  • Förster resonance energy transfer (FRET) paired with fluorescence lifetime imaging microscopy (FLIM)

  • Direct measurement of protein-protein interactions at nanometer scale

  • Quantitative assessment of At1g43140 interactions with SCF components in different cellular compartments

  • Reveals dynamic changes in protein complex formation under various conditions

• Correlative light and electron microscopy (CLEM):

  • Combines immunofluorescence localization with ultrastructural context

  • Enables precise localization of At1g43140 relative to cellular structures

  • Particularly valuable for examining association with membranes or organelles

These advanced imaging applications provide unprecedented insights into At1g43140 function and regulation at subcellular levels beyond conventional microscopy capabilities.

What are the latest developments in computational antibody design applicable to At1g43140 research?

Recent computational antibody design advances offer significant opportunities for At1g43140 research:

• Structure-based antibody design:

  • De novo computational databases containing diverse human-like light and heavy chain combinations can accelerate antibody development

  • CDR redesign utilizing developable antibody frameworks can optimize binding to specific At1g43140 epitopes

  • Computational alanine scanning identifies interfacial residues that significantly contribute to antigen-antibody complex stability

  • These approaches reduce reliance on experimental trial and error for finding initial antibody hits

• AI-powered epitope prediction:

  • Machine learning algorithms trained on antibody-antigen crystal structures predict optimal epitopes

  • Generative adversarial networks create diverse libraries of novel antibodies emulating somatically hypermutated responses

  • These methods can identify immunogenic regions of At1g43140 that differ from other cullin family proteins

• Developability assessment tools:

  • In silico prediction of antibody biophysical properties (stability, solubility) identifies potential manufacturing challenges early

  • Virtual screening of antibody candidates against common developability issues reduces experimental burden

  • These computational solutions represent the most developed application of computational protein design in biotherapeutic discovery

• Integration with experimental validation:

  • Hybrid approaches combining computational prediction with high-throughput experimental validation

  • Rapid iteration between in silico design and experimental testing accelerates development timelines

  • Greater dissemination of successful case studies is needed to raise awareness of these computational capabilities

Implementing these computational approaches can significantly accelerate At1g43140 antibody development while enhancing specificity, particularly for distinguishing between closely related cullin family members.

What are the recommended best practices for At1g43140 antibody validation and usage?

Comprehensive validation and standardized usage protocols are essential for reliable At1g43140 antibody applications:

• Minimum validation criteria:

  • Specificity verification using knockout/knockdown lines or competing peptides

  • Application-specific validation (not assuming Western blot validation transfers to immunohistochemistry)

  • Lot-to-lot consistency testing when replenishing antibody stocks

  • Cross-reactivity assessment against other cullin family proteins

  • Publishing detailed antibody information in methods sections (source, catalog number, RRID, dilution)

• Experimental standardization:

  • Maintain detailed protocols with all buffer compositions, incubation times, and temperatures

  • Include both positive and negative controls in every experiment

  • Pre-adsorb antibodies with plant extracts from At1g43140 knockout lines to reduce background

  • Use consistent image acquisition settings when comparing different samples

  • Apply appropriate normalization for quantitative analyses

• Data reporting requirements:

  • Include representative images of full blots/gels with molecular weight markers

  • Provide raw data and analysis methods in supplementary materials

  • Clearly state any image processing performed (contrast adjustment, background subtraction)

  • Report both technical and biological replication numbers

  • Deposit antibody validation data in public repositories when possible

• Long-term storage considerations:

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Maintain precise records of antibody performance over time

  • Test aged antibodies against freshly purchased lots to detect potential degradation

  • Include preservatives for long-term storage (sodium azide, glycerol)

Following these best practices ensures reproducible research with At1g43140 antibodies and facilitates meaningful comparison of results across different studies and laboratories.

How can researchers troubleshoot common issues with At1g43140 antibodies in different applications?

Systematic troubleshooting approaches can resolve common challenges with At1g43140 antibodies:

• Western blot issues:

  • Weak signal: Increase antibody concentration, extend incubation time, or use signal enhancement systems

  • Multiple bands: Verify specificity with knockout controls, optimize blocking conditions, or perform peptide competition

  • High background: Increase washing duration/stringency, try alternative blocking agents, or purify antibody

  • No detection: Ensure epitope is not destroyed during sample preparation, try alternative extraction methods

• Immunolocalization challenges:

  • Poor signal-to-noise ratio: Optimize fixation method, increase antibody concentration, or try signal amplification

  • Non-specific staining: Extend blocking time, use different blocking agents, or pre-absorb antibody

  • Inconsistent results: Standardize tissue handling, fixation duration, and permeabilization conditions

  • Autofluorescence: Use appropriate quenching methods or switch to non-fluorescent detection

• Immunoprecipitation problems:

  • Low recovery: Increase antibody amount, optimize extraction conditions, or extend incubation time

  • Co-precipitating contaminants: Increase wash stringency, pre-clear lysates, or use crosslinked antibody-bead complexes

  • Degraded target protein: Add additional protease inhibitors, reduce processing time, or perform procedure at lower temperature

• ChIP challenges:

  • Low enrichment: Optimize crosslinking conditions, increase antibody amount, or improve sonication

  • High background: Perform more stringent washes, add competitors like salmon sperm DNA, or optimize blocking

  • Poor reproducibility: Standardize chromatin preparation, increase biological replicates, or use automated systems

For each application, systematic variation of a single parameter at a time while keeping others constant represents the most efficient troubleshooting approach for optimizing At1g43140 antibody performance.

What future research directions will benefit from improved At1g43140 antibody tools?

Advanced At1g43140 antibody tools will enable several promising research directions:

• Functional specialization studies:

  • Comprehensive comparison of At1g43140 versus AtCUL1 tissue-specific expression patterns

  • Identification of unique substrate recognition profiles for different cullin-based E3 ligases

  • Investigation of At1g43140 roles in specific developmental processes or stress responses

  • These studies will address fundamental questions about functional redundancy versus specialization within the cullin family

• Dynamic regulation mechanisms:

  • Real-time monitoring of At1g43140 post-translational modifications

  • Investigation of spatial and temporal regulation of SCF complex assembly

  • Identification of factors controlling At1g43140 subcellular localization

  • These approaches will reveal regulatory mechanisms controlling At1g43140 activity

• Protein interaction networks:

  • Comprehensive mapping of At1g43140-specific protein-protein interactions

  • Comparative interactomics across different tissues, developmental stages, and stress conditions

  • Identification of regulators and substrates unique to At1g43140-containing complexes

  • These studies will position At1g43140 within the broader cellular signaling network

• Translational applications:

  • Engineering plants with modified At1g43140 function for improved stress tolerance

  • Developing crops with altered protein degradation dynamics for enhanced agronomic traits

  • Creating biosensors based on At1g43140 antibodies to monitor plant stress responses

  • These applications could contribute to sustainable agriculture and climate resilience