At2g36090 Antibody

What is AT2G36090 and why is it important for plant research?

AT2G36090 is a protein-coding gene located on chromosome 2 of Arabidopsis thaliana that encodes an F-box family protein according to the Araport11 annotation . F-box proteins are particularly important in plant research as they function as components of SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes, which mediate protein degradation through the ubiquitin-proteasome pathway. This protein degradation system is crucial for numerous cellular processes including hormone signaling, development, and stress responses in plants. Understanding AT2G36090's specific function could provide insights into regulatory mechanisms controlling plant growth, development, and environmental adaptation.

What techniques are available for detecting AT2G36090 protein in plant samples?

Several techniques can be employed to detect AT2G36090 protein in plant tissues:

• Immunoblotting (Western blot): Using specific antibodies against AT2G36090 to detect the protein after separation by SDS-PAGE. This requires high-quality, validated antibodies with confirmed specificity.

• Immunoprecipitation: Enriching the protein from plant extracts using specific antibodies before detection.

• Mass spectrometry: For identification and quantification of AT2G36090 protein in complex samples.

• GFP fusion approaches: Similar to those used for related proteins, where AT2G36090 can be tagged with GFP to visualize its subcellular localization and expression patterns .

• Epitope tagging: Adding small epitope tags (HA, FLAG, etc.) to the protein for detection using commercially available antibodies when specific antibodies are not available.

Each method has specific sample preparation requirements, with immunoblotting being particularly useful for monitoring protein levels across different tissues or conditions.

How should researchers validate the specificity of AT2G36090 antibodies?

Validating antibody specificity for AT2G36090 requires several complementary approaches:

Using T-DNA insertion lines in the AT2G36090 gene as negative controls is particularly important, similar to the approach used for characterizing other Arabidopsis genes .

How can researchers determine the subcellular localization of AT2G36090 protein?

Determining the subcellular localization of AT2G36090 requires multiple complementary approaches:

• Fluorescent protein fusion: Creating N- or C-terminal GFP fusions with AT2G36090 and expressing in Arabidopsis cells to visualize localization by confocal microscopy. This approach has been successfully used for related proteins including kinases in Arabidopsis .

• Immunofluorescence microscopy: Using specific antibodies against AT2G36090 coupled with fluorescently-labeled secondary antibodies to visualize the native protein in fixed plant tissues.

• Cell fractionation: Separating different cellular compartments followed by immunoblotting with AT2G36090 antibodies to determine which fraction contains the protein.

• Co-localization studies: Combining AT2G36090 detection with markers for specific cellular compartments (e.g., nuclei, ER, cytosol) to precisely identify its location.

For optimal results, researchers should use native promoter-driven expression rather than constitutive promoters like 35S, which can sometimes cause artifactual localization patterns. Comparing results across multiple methods provides the most reliable localization data.

What are the recommended approaches for studying AT2G36090 protein-protein interactions?

Several complementary methods can be employed to study AT2G36090 interactions:

• Yeast two-hybrid (Y2H): Screening for interaction partners using AT2G36090 as bait. Particularly useful for identifying potential SCF complex components and substrates.

• Co-immunoprecipitation (Co-IP): Using AT2G36090 antibodies to pull down the protein complex from plant extracts, followed by mass spectrometry to identify interacting partners.

• Bimolecular Fluorescence Complementation (BiFC): Testing specific interactions in planta by fusing AT2G36090 and potential partners to complementary fragments of a fluorescent protein.

• Proximity labeling: Using BioID or APEX2 fusions with AT2G36090 to identify proximal proteins in living cells.

• In vitro pull-down assays: Using recombinant AT2G36090 protein to identify direct binding partners.

The most comprehensive approach would integrate multiple methods, with initial Y2H screens followed by validation using Co-IP and BiFC to confirm interactions in planta. Special attention should be paid to interactions with Arabidopsis Skp1-like proteins (ASKs) and potential substrate proteins, as these would confirm its function in an SCF complex.

How can researchers investigate the role of AT2G36090 in plant development and stress responses?

A comprehensive approach to understanding AT2G36090 function includes:

• Phenotypic analysis of knockout and overexpression lines: Examining plant development, morphology, and responses to various stresses. This approach was effectively used for the neighboring gene AT2G36080 (NGAL1), which showed altered flower petal and shoot development when overexpressed .

• Expression profiling: Determining when and where AT2G36090 is expressed using:

  • Promoter-GUS fusions (similar to methods used for AT2G36080)

  • RT-PCR/qPCR analysis across tissues and conditions

  • Public transcriptome databases

• Identification of degradation targets: Using:

  • Comparative proteomics between wild-type and knockout plants

  • Ubiquitination assays to confirm F-box protein activity

  • Protein stability assays with potential substrates

• Epistasis analysis: Crossing AT2G36090 mutants with mutants of potential pathway components to establish genetic hierarchies.

Creating a tissue-specific or inducible knockout/knockdown system can be particularly valuable if constitutive knockout causes lethality or severe developmental defects.

Why might researchers experience non-specific binding with AT2G36090 antibodies?

Non-specific binding is a common challenge with plant protein antibodies and may occur for several reasons:

Performing parallel experiments with knockout mutant plant materials is crucial for verifying the specificity of any observed signals. Additionally, pre-adsorption of antibodies with recombinant AT2G36090 protein can help identify which bands represent specific binding.

How can researchers optimize protein extraction for better AT2G36090 detection?

Optimizing protein extraction is critical for successfully detecting AT2G36090:

• Buffer optimization:

  • Test multiple extraction buffers (RIPA, Tris-based, phosphate-based)

  • Include 1% Triton X-100 or NP-40 to solubilize membrane-associated proteins

  • Add 5-10 mM DTT or β-mercaptoethanol to reduce disulfide bonds

  • Include protease inhibitor cocktail with specific inhibitors for plant proteases

• Extraction conditions:

  • Maintain cold temperatures throughout extraction (4°C)

  • Consider flash-freezing tissues in liquid nitrogen before grinding

  • Use mechanical disruption (bead beating for 30-60 seconds) for efficient lysis

• Sample concentration:

  • Use TCA precipitation or acetone precipitation to concentrate proteins

  • Consider immunoprecipitation to enrich for AT2G36090 before analysis

• Tissue selection:

  • Analyze expression data to identify tissues with highest AT2G36090 expression

  • Compare protein levels across different developmental stages

Similar approaches have been successfully employed in studies of other Arabidopsis proteins, such as the analysis of kinase proteins described in the Lamberti study .

What controls are essential when using AT2G36090 antibodies in immunoprecipitation experiments?

When performing immunoprecipitation with AT2G36090 antibodies, several controls are essential:

• Negative controls:

  • IP from knockout/mutant plants lacking AT2G36090

  • IP using non-specific IgG of the same species as the AT2G36090 antibody

  • Pre-immune serum control if using custom antibodies

• Input control:

  • Reserve 5-10% of the extract before IP to confirm presence of target protein

• Blocked antibody control:

  • Perform parallel IP with antibody pre-incubated with immunizing peptide

• Denaturing vs. native conditions:

  • Compare IPs under different conditions to optimize for protein complex preservation

• Validation by mass spectrometry:

  • Confirm identity of immunoprecipitated proteins by peptide mass fingerprinting

These controls help distinguish true AT2G36090 interactions from non-specific binding or contaminants, increasing confidence in the results and facilitating publication in high-impact journals.

What alternatives exist when specific antibodies for AT2G36090 are unavailable or ineffective?

When specific antibodies prove challenging, researchers can employ several alternative approaches:

• Epitope tagging strategies:

  • Generate transgenic plants expressing AT2G36090 fused to epitope tags (HA, FLAG, cMyc)

  • Use CRISPR/Cas9 to add an endogenous tag to the native gene

  • Express tagged protein under native promoter control to maintain physiological expression levels

• Proximity labeling:

  • Fuse AT2G36090 to BioID or APEX2 enzymes that biotinylate nearby proteins

  • Detect the fusion protein using commercial anti-biotin antibodies or streptavidin

• MS-based proteomics:

  • Use targeted proteomics (SRM/MRM) to detect specific AT2G36090 peptides

  • Employ label-free quantification to measure relative abundance across samples

• Activity-based assays:

  • Develop assays based on the predicted F-box protein function in protein degradation

  • Monitor ubiquitination activity in the presence/absence of AT2G36090

A combined approach utilizing both tagging strategies and functional assays provides the most comprehensive characterization of AT2G36090 in the absence of specific antibodies.

How can researchers analyze post-translational modifications of AT2G36090?

Analysis of post-translational modifications (PTMs) requires specialized approaches:

• Phosphorylation analysis:

  • Immunoprecipitation followed by phospho-specific antibody detection

  • Phospho-enrichment using TiO₂ or IMAC followed by mass spectrometry

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

  • In vitro kinase assays with candidate kinases, similar to those used in STY kinase studies

• Ubiquitination analysis:

  • Detection using anti-ubiquitin antibodies after AT2G36090 immunoprecipitation

  • Tandem ubiquitin binding entity (TUBE) pulldowns followed by AT2G36090 detection

  • Mass spectrometry to identify ubiquitination sites

• Other modification analyses:

  • SUMOylation, acetylation, and methylation can be analyzed using modification-specific antibodies

  • Mass spectrometry with specific enrichment strategies for each modification type

These analyses are particularly relevant for F-box proteins, which often undergo regulatory PTMs that affect their stability and substrate recognition abilities.

How can researchers integrate AT2G36090 studies with systems biology approaches?

Integrating AT2G36090 research with systems biology requires:

• Transcriptome integration:

  • Correlate AT2G36090 expression with global gene expression patterns

  • Identify co-expressed genes that may function in similar pathways

  • Compare expression profiles between wild-type and AT2G36090 mutant plants

• Proteome-wide interactions:

  • Map AT2G36090 into protein interaction networks

  • Predict functional relationships based on interaction partners

  • Identify condition-specific interactions under different stresses

• Metabolomics integration:

  • Analyze metabolite profiles in AT2G36090 mutants

  • Identify metabolic pathways affected by AT2G36090 function

• Multi-omics data integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Use pathway analysis tools to identify enriched biological processes

  • Apply machine learning to predict AT2G36090 function from integrated datasets

This systems-level understanding places AT2G36090 in the broader context of plant cellular processes, potentially revealing unexpected roles or connections to known signaling pathways.

How might structural biology approaches enhance understanding of AT2G36090 function?

Structural biology can provide crucial insights into AT2G36090 function:

• Protein structure prediction:

  • Use AlphaFold2 or RoseTTAFold to predict AT2G36090 structure

  • Identify key structural features and potential substrate binding sites

  • Compare structural predictions with known F-box protein structures

• Experimental structure determination:

  • X-ray crystallography of recombinant AT2G36090 alone or in complex with SCF components

  • Cryo-EM analysis of the entire SCF complex containing AT2G36090

  • NMR studies of specific domains for dynamic information

• Structure-guided functional studies:

  • Site-directed mutagenesis of predicted key residues

  • Design of specific peptide inhibitors based on structural information

  • Computational docking of potential substrates

Understanding the structural basis of AT2G36090 substrate recognition would significantly advance our knowledge of its biological function and potentially enable the design of specific inhibitors or activators.

What considerations are important when analyzing AT2G36090 expression across different environmental conditions?

Environmental regulation of AT2G36090 expression requires careful experimental design:

• Experimental conditions:

  • Precisely control and document growth conditions (light, temperature, humidity)

  • Apply standardized stress treatments with appropriate controls

  • Consider diurnal regulation and sample at multiple time points

• Expression analysis methods:

  • RT-qPCR with validated reference genes stable under test conditions

  • Western blot with loading controls appropriate for stress conditions

  • Promoter-reporter fusions to visualize spatial expression patterns

• Data interpretation challenges:

  • Distinguish direct vs. indirect effects of environmental conditions

  • Account for developmental differences between control and stressed plants

  • Consider post-transcriptional regulation that may affect protein levels

• Replication and validation:

  • Biological replicates from independent experiments

  • Technical replicates to ensure measurement reliability

  • Validation using multiple detection methods

These considerations help ensure that observed changes in AT2G36090 expression are genuinely linked to the environmental conditions being tested rather than experimental artifacts.