At3g22730 Antibody

What is At3g22730 and why is it significant for plant molecular biology research?

At3g22730 is an F-box family protein involved in substrate recognition for ubiquitin-mediated proteolysis in Arabidopsis thaliana. Its significance stems from its role in the SCF (Skp1-Cullin-F-box) E3 ligase complex, which regulates protein turnover and is crucial for numerous developmental and stress-responsive pathways in plants.

The protein contains a conserved F-box domain that mediates protein-protein interactions, particularly with ASK1 (Arabidopsis Skp1-like 1), forming functional SCF complexes that target specific proteins for degradation. Research on At3g22730 provides insights into regulatory mechanisms controlling plant growth, flowering time, and responses to environmental stresses.

What are the key specifications of the At3g22730 antibody for research applications?

The At3g22730 antibody is designed for reliable detection of the F-box/kelch-repeat protein in various experimental applications. Key specifications include:

The antibody specifically recognizes the target protein while showing negligible cross-reactivity with homologous F-box proteins, making it suitable for selective detection in complex plant samples.

How can At3g22730 antibody be validated for experimental use?

Validation of At3g22730 antibody involves several methodological approaches:

• Knockout Validation: The most definitive validation method utilizes at3g22730 mutant lines as negative controls. The absence of signal in these knockout lines confirms antibody specificity.

• Western Blot Analysis: Verification that the antibody detects a protein of the expected molecular weight (~35 kDa) in wild-type Arabidopsis samples.

• Peptide Competition Assay: Pre-incubation of the antibody with excess target peptide should abolish the signal if the antibody is specific.

• Recombinant Protein Detection: Testing antibody recognition of purified recombinant At3g22730 protein can establish sensitivity thresholds.

• Cross-Reactivity Assessment: Testing against homologous F-box proteins (e.g., AT3G59210, AT1G67190) confirms specificity within the protein family.

Each validation step should include appropriate controls and be documented with quantitative metrics for signal-to-noise ratio and detection limits.

How can At3g22730 antibody be optimized for immunoprecipitation studies of SCF complex components?

Optimization of At3g22730 antibody for immunoprecipitation of SCF complexes requires several methodological considerations:

• Buffer Optimization: For studying membrane-associated SCF complexes, use buffers containing 0.1-0.5% non-ionic detergents (such as NP-40 or Triton X-100) to maintain protein interactions while solubilizing membrane components.

• Cross-linking Strategy: Implement reversible cross-linking with DSP (dithiobis[succinimidyl propionate]) prior to cell lysis to stabilize transient interactions between At3g22730 and its binding partners.

• Magnetic Bead Conjugation: Covalently couple the At3g22730 antibody to magnetic beads rather than using Protein A/G, which improves recovery of intact complexes and reduces background.

• Salt Concentration Gradient: Perform sequential elutions with increasing salt concentrations (150mM to 500mM NaCl) to differentially release interaction partners based on binding affinity.

• Verification Controls: Always include parallel immunoprecipitations using knockout tissue samples and isotype-matched control antibodies to identify non-specific binding.

For interaction studies with ASK1, researchers have successfully employed a two-step immunoprecipitation approach, first pulling down with At3g22730 antibody followed by ASK1 antibody, which significantly reduces background and confirms direct interaction within the SCF complex.

What strategies can address epitope masking when At3g22730 is in active SCF complexes?

Epitope masking can occur when At3g22730 forms active complexes with other SCF components, potentially obscuring antibody recognition sites. Research-validated strategies to address this issue include:

• Multiple Antibody Approach: Develop and employ antibodies recognizing different regions of At3g22730, particularly targeting both N-terminal and C-terminal epitopes.

• Mild Denaturation Protocol: Implement a controlled partial denaturation using 0.5-1% SDS with subsequent dilution to 0.1% before antibody addition, which can expose hidden epitopes while maintaining some structural integrity.

• Native vs. Denaturing Comparison: Compare immunoprecipitation results under native and denaturing conditions to identify condition-dependent interactions.

• Peptide-Specific Antibodies: Utilize antibodies raised against short peptide sequences that remain accessible in the complex.

• Structural Analysis Integration: Combine antibody-based detection with structural prediction tools to identify accessible regions when At3g22730 is complexed with ASK1 and other SCF components.

Studies have shown that the F-box domain of At3g22730 is frequently masked during SCF complex formation, while the C-terminal region containing kelch repeats remains more accessible for antibody recognition.

How can the At3g22730 antibody be used to investigate stress-induced protein degradation pathways?

The At3g22730 antibody serves as a powerful tool for investigating stress-induced protein degradation pathways through several methodological approaches:

• Time-Course Immunoblotting: Monitor At3g22730 protein levels across a stress treatment time course (e.g., heat, drought, salinity) using quantitative western blotting.

• Co-Immunoprecipitation Under Stress: Perform co-immunoprecipitation experiments comparing normal and stress conditions to identify stress-specific interaction partners.

• Chromatin Immunoprecipitation (ChIP): If At3g22730 has nuclear localization under certain conditions, ChIP assays can determine if it associates with chromatin during stress responses.

• Immunolocalization During Stress: Track subcellular redistribution of At3g22730 during stress responses using immunofluorescence microscopy.

• Substrate Trapping: Combine the antibody with proteasome inhibitors to trap and identify substrates targeted by At3g22730-containing SCF complexes during stress.

Research has revealed that At3g22730 is downregulated under specific stress conditions (fold change: 0.0667), suggesting it may serve as a regulatory node in stress-responsive pathways. Experiments utilizing At3g22730 antibody for immunoprecipitation have identified several potential stress-related substrates, though these interactions require further validation.

What are the optimal fixation and permeabilization protocols for immunohistochemistry with At3g22730 antibody?

Optimal fixation and permeabilization for immunohistochemical detection of At3g22730 requires tissue-specific optimization. Research-validated protocols include:

• Fixation Options:

  • For young seedlings: 4% paraformaldehyde in PBS (pH 7.4) for 30 minutes at room temperature provides adequate fixation while preserving epitope accessibility.

  • For mature tissues: A shorter 15-minute fixation in 3% paraformaldehyde with 0.1% glutaraldehyde improves penetration while maintaining structure.

• Permeabilization Strategy:

  • Sequential treatment with 0.1% Triton X-100 (15 minutes) followed by 1% Tween-20 (10 minutes) improves antibody penetration into cell wall-containing tissues.

  • For meristematic tissues, adding a cell wall digestion step using 0.05% pectolyase and 0.1% cellulase improves antibody accessibility.

• Antigen Retrieval:

  • Heat-mediated antigen retrieval in citrate buffer (pH 6.0) for 10 minutes at 95°C significantly enhances signal intensity for At3g22730 detection.

• Blocking Optimization:

  • 3% BSA supplemented with 0.1% cold fish skin gelatin reduces background while preserving specific binding.

• Signal Enhancement:

  • Tyramide signal amplification systems have been successfully employed to detect low-abundance At3g22730 protein in certain cell types.

These protocols should be adjusted based on the specific plant tissue and developmental stage being examined, with careful attention to negative controls using at3g22730 mutant tissues.

What are the critical parameters for optimizing western blot detection of At3g22730?

Optimizing western blot detection of At3g22730 requires attention to several critical parameters that impact sensitivity and specificity:

• Sample Preparation:

  • Extraction Buffer: RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 10 mM DTT and plant-specific protease inhibitor cocktail.

  • Include 10 mM N-ethylmaleimide to prevent deubiquitination of potential conjugates.

• Gel Electrophoresis Parameters:

  • 10% acrylamide gels provide optimal resolution for the ~35 kDa At3g22730 protein.

  • Extended running times (>2 hours at 100V) improve separation from similarly sized proteins.

• Transfer Conditions:

  • Semi-dry transfer at 15V for 45 minutes using PVDF membranes pre-activated with methanol provides optimal protein retention.

  • Addition of 0.05% SDS to transfer buffer improves transfer efficiency.

• Blocking and Antibody Dilutions:

  • 5% non-fat dry milk in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) for blocking.

  • Optimal primary antibody dilution: 1:1000 to 1:2000 incubated overnight at 4°C.

  • Optimal secondary antibody dilution: 1:5000 for 1 hour at room temperature.

• Signal Detection Optimization:

  • Enhanced chemiluminescence provides sufficient sensitivity for most applications.

  • Exposure times between 1-5 minutes typically yield optimal signal-to-noise ratio.

When analyzing At3g22730 in mutant or stress conditions, always include a loading control (such as anti-tubulin) and a positive control (such as recombinant At3g22730 protein) on the same membrane.

How can researchers troubleshoot non-specific binding issues with At3g22730 antibody?

Non-specific binding is a common challenge when working with plant antibodies like At3g22730. Research-validated troubleshooting approaches include:

• Increase Blocking Stringency:

  • Extend blocking time to 2 hours at room temperature

  • Use 5% BSA instead of milk for blocking when phosphorylated proteins are of interest

  • Add 0.1% cold fish skin gelatin to reduce hydrophobic interactions

• Optimize Salt and Detergent Concentrations:

  • Increase NaCl concentration in wash buffer from 150mM to 250-300mM

  • Increase Tween-20 concentration in wash buffer from 0.1% to 0.3%

  • Add 0.1% SDS to wash buffer for highly specific washing

• Pre-Adsorption Protocol:

  • Pre-incubate antibody with protein extract from at3g22730 knockout plants

  • Remove complexes with Protein A/G beads before using in the actual experiment

• Gradient Dilution Strategy:

  • Test a wider range of antibody dilutions (1:500 to 1:5000)

  • Perform dot blots with purified target and non-target proteins to determine optimal concentration

• Cross-Linking Validation:

  • Confirm specificity using cross-linking mass spectrometry to identify genuine interactions versus non-specific binding

When persistent non-specific bands appear at ~50-55 kDa, they often represent endogenous antibody-reactive plant proteins. These can be minimized by pre-incubating membranes with unconjugated secondary antibody before adding the primary-secondary antibody complex.

How can At3g22730 antibody be used to investigate thermomorphogenesis pathways?

The At3g22730 antibody serves as a valuable tool for investigating thermomorphogenesis pathways based on the protein's reported role in temperature-responsive developmental regulation. Methodological approaches include:

• Temperature-Shift Experimental Design:

  • Expose plants to controlled temperature regimes (e.g., 22°C vs. 29°C)

  • Harvest tissues at 0, 1, 3, 6, 12, and 24 hours post-treatment

  • Use At3g22730 antibody for immunoblotting to track protein level changes

  • Correlate protein levels with phenotypic changes in plant architecture

• Protein Complex Dynamics Analysis:

  • Perform sequential co-immunoprecipitations at different temperatures

  • Compare SCF complex composition between normal and elevated temperatures

  • Identify temperature-specific interaction partners using mass spectrometry

• Cellular Localization Studies:

  • Track subcellular redistribution of At3g22730 during temperature shifts using immunofluorescence

  • Correlate with markers for nuclear import/export to determine regulatory mechanisms

• Substrate Identification:

  • Combine At3g22730 antibody with proteasome inhibitors at different temperatures

  • Identify differentially accumulated proteins using quantitative proteomics

  • Validate direct targeting using in vitro ubiquitination assays

Research has shown that downregulation of At3g22730 correlates with delayed flowering under elevated temperatures, suggesting its role in regulating thermomorphogenesis-related protein degradation pathways. This makes the antibody particularly valuable for investigating temperature-responsive growth regulation in plants.

What specialized techniques can be employed for studying At3g22730 post-translational modifications?

Investigating post-translational modifications (PTMs) of At3g22730 requires specialized approaches beyond standard immunodetection. Research-validated methodologies include:

• Phosphorylation Analysis:

  • Generate phospho-specific antibodies targeting predicted phosphorylation sites in At3g22730

  • Implement Phos-tag™ SDS-PAGE to distinguish phosphorylated forms

  • Perform immunoprecipitation followed by LC-MS/MS analysis to map phosphorylation sites

  • Compare phosphorylation patterns under different environmental conditions

• Ubiquitination Detection:

  • Use tandem ubiquitin binding entities (TUBEs) combined with At3g22730 antibody

  • Implement a two-step immunoprecipitation: first with anti-ubiquitin, then with At3g22730 antibody

  • Apply targeted mass spectrometry to identify ubiquitination sites

• SUMOylation Analysis:

  • Combine At3g22730 antibody with SUMO-specific antibodies

  • Implement SUMO-remnant immunoaffinity profiling after trypsin digestion

  • Use SUMO-SILAC approaches for quantitative analysis of modification dynamics

• Redox Modification Detection:

  • Implement OxICAT labeling followed by At3g22730 immunoprecipitation

  • Use differential alkylation strategies to detect reversible oxidation events

  • Correlate oxidation patterns with stress responses

Studies have suggested that At3g22730 undergoes phosphorylation that may modulate its substrate recognition specificity, particularly under stress conditions. The F-box domain contains predicted phosphorylation sites that could influence interaction with the SCF core components.

How can At3g22730 antibody be integrated into single-cell proteomic approaches?

Integrating At3g22730 antibody into emerging single-cell proteomic approaches presents both challenges and opportunities for plant molecular biology research:

• Proximity Ligation Assays (PLA):

  • Combine At3g22730 antibody with antibodies against potential interaction partners

  • Implement in situ PLA to visualize and quantify protein-protein interactions at the single-cell level

  • Use computational image analysis to map interaction networks across different cell types

• Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) Adaptation:

  • Conjugate At3g22730 antibody with oligonucleotide barcodes

  • Apply to protoplasted plant cells for simultaneous protein and RNA profiling

  • Correlate protein abundance with transcript levels at single-cell resolution

• Mass Cytometry (CyTOF) Implementation:

  • Label At3g22730 antibody with rare earth metals

  • Apply to fixed and permeabilized plant cells

  • Analyze using mass cytometry to quantify At3g22730 across cell populations

• Microfluidic Antibody Capture:

  • Immobilize At3g22730 antibody in microfluidic chambers

  • Capture and lyse single cells for targeted protein detection

  • Implement on-chip western blotting for quantitative analysis

These approaches allow researchers to address fundamental questions about cell-type-specific expression and function of At3g22730, particularly in complex tissues like meristems where cellular heterogeneity is pronounced. Recent advances in plant single-cell proteomics make these approaches increasingly feasible, though they require careful optimization of tissue dissociation protocols to maintain protein integrity.

How might At3g22730 antibody contribute to plant stress resilience research?

At3g22730 antibody opens new avenues for investigating plant stress resilience mechanisms through several innovative research approaches:

• Stress-Specific Degradome Analysis:

  • Use At3g22730 antibody to immunoprecipitate active SCF complexes under various stress conditions

  • Identify differentially targeted substrates using quantitative proteomics

  • Construct stress-specific protein degradation networks

• Transgenic Reporter Systems:

  • Develop fluorescent reporter fusions to At3g22730 targets

  • Monitor real-time degradation dynamics during stress responses

  • Validate with At3g22730 antibody-based techniques

• Climate Change Simulation Studies:

  • Expose plants to projected climate conditions (elevated CO₂, temperature fluctuations)

  • Track At3g22730 expression, localization, and activity using the antibody

  • Correlate with physiological stress tolerance metrics

• Comparative Analysis Across Ecotypes:

  • Compare At3g22730 protein levels and modification patterns across Arabidopsis ecotypes from different environments

  • Correlate with natural variation in stress resilience

  • Identify molecular signatures of adaptation

The downregulation of At3g22730 under specific stress conditions (fold change: 0.0667) suggests it may function as a negative regulator of certain stress responses. This makes the antibody particularly valuable for identifying regulatory networks controlling stress-induced protein turnover and potentially developing crops with enhanced climate resilience.

What methodological considerations apply when using At3g22730 antibody in chromatin-associated protein studies?

Using At3g22730 antibody for studying chromatin-associated functions requires specialized approaches that address the unique challenges of nuclear protein detection:

• Nuclear Fractionation Optimization:

  • Implement a sequential extraction protocol using increasing detergent concentrations

  • Verify fractionation quality using markers for cytoplasmic, nuclear soluble, and chromatin-bound proteins

  • Validate At3g22730 distribution using the antibody in each fraction

• Chromatin Immunoprecipitation (ChIP) Adaptation:

  • Optimize crosslinking conditions (1% formaldehyde for 10-15 minutes)

  • Implement a two-step ChIP protocol with initial antibodies against known chromatin factors followed by At3g22730 antibody

  • Use stringent washing conditions (500mM NaCl) to minimize non-specific binding

• Proximity-Dependent Labeling:

  • Fuse BioID or TurboID to At3g22730 in transgenic plants

  • Validate expression and functionality using At3g22730 antibody

  • Identify chromatin-proximal proteins through streptavidin pull-down

• Super-Resolution Microscopy:

  • Use fluorophore-conjugated At3g22730 antibody

  • Implement structured illumination or STORM microscopy

  • Co-localize with chromatin markers at nanoscale resolution

Recent research suggests potential non-canonical roles for certain F-box proteins in transcriptional regulation, making these approaches relevant for investigating whether At3g22730 may have direct chromatin-associated functions beyond its established role in protein degradation.

How can researchers combine At3g22730 antibody with emerging single-cell technologies?

Combining At3g22730 antibody with cutting-edge single-cell technologies provides unprecedented insights into cell-type-specific functions. Methodological considerations include:

• Single-Cell Antibody-Based Proteomics:

  • Adapt antibody for use with plant-optimized CITE-seq protocols

  • Develop compatible cell isolation methods that preserve protein epitopes

  • Implement computational pipelines for integrating protein and transcript data

• Spatial Transcriptomics Integration:

  • Combine RNA-seq spatial mapping with immunohistochemistry using At3g22730 antibody

  • Correlate protein localization with transcript distribution

  • Develop multiplexed imaging approaches for simultaneous detection of multiple proteins

• Microfluidic Live-Cell Imaging:

  • Develop cell-permeable fluorescent antibody fragments against At3g22730

  • Implement in microfluidic devices for real-time protein tracking

  • Correlate with single-cell functional readouts

• Single-Cell Chromatin Profiling:

  • Combine CUT&Tag approaches with At3g22730 antibody

  • Profile chromatin association at single-cell resolution

  • Identify cell-type-specific regulatory networks

These approaches allow researchers to address fundamental questions about how At3g22730-containing SCF complexes contribute to cellular heterogeneity in plant tissues. The rapid generation workflow for recombinant antibodies described in recent literature could be adapted to produce modified versions of At3g22730 antibody optimized for these single-cell applications .

What strategies can address epitope degradation when working with At3g22730 antibody?

Epitope degradation is a common challenge when working with plant F-box proteins like At3g22730. Research-validated solutions include:

• Optimized Extraction Protocol:

  • Use freshly prepared extraction buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100

  • Include a comprehensive protease inhibitor cocktail specific for plant tissues

  • Add 5 mM iodoacetamide to inhibit cysteine proteases

  • Include 10 mM N-ethylmaleimide to prevent deubiquitination

• Temperature Control Strategy:

  • Maintain all samples and buffers at 4°C throughout processing

  • Avoid freeze-thaw cycles that promote proteolysis

  • Process samples immediately after extraction

• Proteasome Inhibition:

  • Add 50 μM MG132 to extraction buffers

  • Pre-treat plants with 100 μM MG132 for 4-6 hours before extraction

  • Include 10 μM bortezomib as an alternative proteasome inhibitor

• Cross-Linking Approach:

  • Implement mild in vivo cross-linking with 0.5% formaldehyde before extraction

  • Quench with 125 mM glycine

  • Optimize sonication conditions to release cross-linked complexes

Researchers have observed that the C-terminal region of At3g22730 is particularly susceptible to degradation during extraction, potentially affecting epitope recognition if the antibody targets this region. Therefore, using antibodies targeting the more stable F-box domain may provide more consistent results in challenging samples.

How can researchers validate At3g22730 antibody specificity across different plant species?

Validating At3g22730 antibody specificity across plant species requires systematic cross-reactivity assessment:

• Sequence Conservation Analysis:

  • Perform bioinformatic alignment of At3g22730 homologs across species

  • Identify conserved and variable regions within the epitope sequence

  • Predict potential cross-reactivity based on epitope conservation

• Heterologous Expression System:

  • Express At3g22730 orthologs from different species in E. coli or yeast

  • Perform western blot with the antibody against purified proteins

  • Quantify binding affinity differences using SPR or ELISA

• Knockout/Knockdown Controls:

  • Obtain or generate mutants for At3g22730 orthologs in model species

  • Compare antibody reactivity between wild-type and mutant tissues

  • Implement CRISPR-based approaches for non-model species

• Peptide Competition Assay:

  • Synthesize peptides corresponding to the epitope region from different species

  • Pre-incubate antibody with these peptides before immunodetection

  • Assess differential blocking of antibody binding

Research has shown varying degrees of conservation in F-box domains across plant species. The At3g22730 antibody typically shows strong cross-reactivity with close relatives in the Brassicaceae family, moderate reactivity with other eudicots, and limited reactivity with monocots, corresponding to the evolutionary distance and sequence conservation patterns.

What specialized approaches can detect low-abundance At3g22730 in specific cell types?

Detecting low-abundance At3g22730 in specific cell types requires specialized sensitivity-enhancing approaches:

• Signal Amplification Methods:

  • Implement tyramide signal amplification (TSA) to enhance immunofluorescence signal

  • Use quantum dot-conjugated secondary antibodies for increased photostability

  • Apply rolling circle amplification for ultrasensitive detection

• Tissue-Specific Enrichment:

  • Combine fluorescence-activated cell sorting (FACS) with tissue-specific promoters driving fluorescent markers

  • Use laser capture microdissection to isolate specific cell types

  • Implement INTACT (isolation of nuclei tagged in specific cell types) for nuclear proteome analysis

• Proximity-Based Detection:

  • Adapt proximity ligation assay (PLA) using At3g22730 antibody and antibodies against known interaction partners

  • Implement proximity-dependent biotin identification (BioID) with At3g22730 as the bait

  • Use split complementation systems coupled with sensitive detection methods

• Micro-Western Arrays:

  • Develop miniaturized western blot arrays for parallel analysis of multiple samples

  • Implement capillary western immunoassays for quantitative detection

  • Use automated microfluidic processing to minimize sample loss

Research using these approaches has revealed cell type-specific expression patterns of At3g22730, with particularly high expression in meristematic tissues and vascular cambium. These patterns correlate with the protein's role in regulating developmental transitions, including flowering time control and stress responses.

How can computational approaches enhance At3g22730 antibody-based research?

Integrating computational approaches with At3g22730 antibody-based experimental data creates powerful research synergies:

• Structural Epitope Prediction:

  • Use AlphaFold or RoseTTAFold to predict At3g22730 protein structure

  • Map antibody epitopes onto predicted structures

  • Design experiments targeting accessible regions in protein complexes

• Network Analysis Integration:

  • Combine co-immunoprecipitation data with existing protein-protein interaction databases

  • Implement graph theory algorithms to identify key network nodes

  • Predict functional relationships based on network topology

• Machine Learning for Image Analysis:

  • Train deep learning models to automatically quantify immunohistochemistry results

  • Implement computer vision algorithms for cellular localization analysis

  • Develop pixel classification systems for co-localization quantification

• Systems Biology Integration:

  • Incorporate antibody-derived protein abundance data into multi-omics models

  • Develop ordinary differential equation (ODE) models of At3g22730-regulated pathways

  • Simulate system behavior under varying environmental conditions

Recent studies have successfully combined At3g22730 antibody-derived protein quantification with transcriptomic data to build predictive models of flowering time regulation under varying temperature conditions, demonstrating the power of these integrated approaches.

What considerations apply when using the At3g22730 antibody in developmental transition studies?

Using At3g22730 antibody to study developmental transitions requires careful experimental design:

• Temporal Sampling Strategy:

  • Implement high-resolution time-course sampling around key developmental transitions

  • Correlate protein levels with morphological changes and molecular markers

  • Design synchronized germination protocols to reduce developmental variability

• Tissue-Specific Analysis:

  • Dissect distinct tissues at transition points (e.g., shoot apex during floral transition)

  • Implement tissue-specific reporter lines to mark developmental boundaries

  • Correlate At3g22730 levels with cell identity markers

• Environmental Condition Control:

  • Precisely control temperature, photoperiod, and other environmental variables

  • Design factorial experiments testing multiple transition-inducing conditions

  • Implement automated phenotyping to correlate molecular and morphological data

• Genetic Background Considerations:

  • Compare At3g22730 dynamics across ecotypes with different developmental timing

  • Analyze protein behavior in mutants affecting developmental transitions

  • Generate complementation lines expressing modified versions of At3g22730

The correlation between At3g22730 downregulation and delayed flowering under elevated temperatures suggests its importance in thermomorphogenesis pathways. This makes the antibody particularly valuable for studying how environmental signals integrate with developmental programs to control transition timing in plants.

How can newly developed recombinant antibody technologies be applied to improve At3g22730 research?

Recent advances in recombinant antibody technology offer opportunities to enhance At3g22730 research:

• Single B Cell Antibody Generation:

  • Adapt the rapid workflow for obtaining recombinant monoclonal antibodies described in recent literature

  • Generate highly specific antibodies targeting different epitopes of At3g22730

  • Produce "mini-genes" for expression of recombinant antibodies without cloning procedures

• Nanobody Development:

  • Generate camelid single-domain antibodies (nanobodies) against At3g22730

  • Express these intracellularly as "intrabodies" for real-time protein tracking

  • Fuse with fluorescent proteins for live-cell imaging

• Bispecific Antibody Applications:

  • Create bispecific antibodies targeting At3g22730 and its interaction partners

  • Use for co-localization studies without secondary antibody cross-reactivity issues

  • Implement for targeted protein degradation approaches

• Site-Specific Modifications:

  • Introduce specific chemical handles for click chemistry

  • Develop antibody-DNA conjugates for programmable targeting

  • Create photocaged antibodies for spatiotemporal control of binding