At3g18150 Antibody

What is At3g18150 and why is it important in plant research?

At3g18150 is a gene locus in Arabidopsis thaliana that encodes a putative F-box/LRR-repeat protein belonging to the RNI-like superfamily . This protein plays significant roles in plant stress responses and developmental processes. F-box proteins are important components of SCF (Skp1-Cullin-F-box) complexes that mediate protein degradation via the ubiquitin-proteasome pathway, making At3g18150 particularly relevant for studying plant signaling networks and stress adaptation mechanisms.

The protein is a downstream target of WRKY75 transcription factor , suggesting its involvement in phosphate starvation responses and potentially in other stress-responsive pathways. Studying At3g18150 contributes to our understanding of how plants regulate protein turnover during development and in response to environmental stimuli.

What types of antibodies are available for At3g18150 protein detection?

While specific commercial At3g18150 antibodies are not explicitly listed in the search results, researchers can employ several approaches for antibody development against this protein:

• Polyclonal antibodies: Generated by immunizing rabbits with purified recombinant At3g18150 protein or synthetic peptides derived from unique regions of the protein sequence .

• Monoclonal antibodies: Produced through hybridoma technology using mice immunized with At3g18150 protein, offering higher specificity but more complex development .

• Recombinant antibodies: Engineered through gene synthesis and expression systems, allowing for customization of binding properties and production without animal immunization .

The selection depends on research requirements, with polyclonal antibodies offering broader epitope recognition but monoclonal antibodies providing consistent lot-to-lot reproducibility.

How can I validate the specificity of an At3g18150 antibody?

Validating antibody specificity for At3g18150 requires multiple complementary approaches:

• Western blot analysis: Using wild-type Arabidopsis extracts alongside At3g18150 knockout/knockdown mutants to confirm the absence of signal in mutant lines.

• Recombinant protein controls: Testing the antibody against purified recombinant At3g18150 protein and unrelated plant proteins to assess cross-reactivity.

• Immunoprecipitation followed by mass spectrometry: Confirming that the antibody pulls down the correct protein.

• Pre-absorption tests: Incubating the antibody with excess antigen before immunodetection to verify signal elimination.

• Tissue specificity correlation: Comparing antibody signal pattern with known mRNA expression patterns of At3g18150.

A properly validated antibody should recognize a protein of approximately the predicted molecular weight of At3g18150 protein (~34 kDa, though post-translational modifications may alter migration) with minimal cross-reactivity to other proteins.

What are the optimal methods for extracting and preserving At3g18150 protein for immunodetection?

Optimal extraction of At3g18150 (F-box/LRR-repeat protein) requires protocols designed to preserve protein integrity while maximizing yield:

• Buffer composition: Use a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100 or 0.5% NP-40

  • 10% glycerol

  • 1 mM EDTA

  • Freshly added protease inhibitor cocktail

  • 1 mM DTT or 5 mM β-mercaptoethanol

• Extraction procedure:

  • Grind plant tissue in liquid nitrogen to fine powder

  • Add 3-5 volumes of extraction buffer

  • Homogenize thoroughly

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

  • Collect supernatant while avoiding lipid layer

• Preservation methods:

  • For short-term storage (1-2 weeks): Store at -80°C in single-use aliquots

  • For immunoprecipitation: Process immediately after extraction

  • Add 5-10% glycerol as cryoprotectant

  • Avoid repeated freeze-thaw cycles

For fractionation approaches, consider that F-box proteins may associate with multiple cellular compartments depending on their interaction status with SCF complexes.

What dilutions and conditions are typically optimal for At3g18150 antibody in Western blot applications?

While specific dilutions for At3g18150 antibodies aren't standardized, optimal conditions can be determined based on similar plant protein antibodies:

Always perform titration experiments to determine optimal antibody concentration for your specific experimental system . For Western blots using plant samples, consider additional steps:

• Include 1% polyvinylpyrrolidone (PVP) and 2% polyvinylpolypyrrolidone (PVPP) in extraction buffer to remove phenolic compounds

• Use PVDF membranes rather than nitrocellulose for improved protein retention

• Extend blocking time to 2 hours to reduce background in plant samples

How can I optimize immunoprecipitation protocols for studying At3g18150 protein interactions?

Optimizing immunoprecipitation (IP) for At3g18150 protein interactions requires consideration of F-box protein dynamics:

• Crosslinking approach:

  • Treat plant tissue with 1% formaldehyde for 10 minutes to preserve transient interactions

  • Quench with 125 mM glycine

  • This preserves interactions with SCF complex components and potential substrates

• IP buffer optimization:

  • Test different salt concentrations (100-300 mM NaCl)

  • Evaluate detergent types and concentrations (0.1-1% NP-40, 0.1-0.5% Triton X-100)

  • Include 10-20 mM MG132 (proteasome inhibitor) to stabilize substrate interactions

• Antibody coupling:

  • Covalently couple purified At3g18150 antibody to Protein A/G beads using dimethyl pimelimidate

  • Use 5-10 μg antibody per IP reaction

  • Pre-clear lysates with uncoupled beads to reduce non-specific binding

• Sequential IP for complex purification:

  • First IP with At3g18150 antibody

  • Elute under mild conditions (peptide competition)

  • Second IP with antibody against predicted interactor

• Controls:

  • IgG isotype control

  • Immunoprecipitation from knockout/knockdown lines

  • Pre-absorption of antibody with antigen

This approach will help identify both stable SCF complex components and transient substrate interactions.

How can At3g18150 antibodies be used to study protein localization changes during plant stress responses?

At3g18150 protein localization can be dynamically regulated during stress responses, and antibody-based approaches offer several strategies to monitor these changes:

• Subcellular fractionation combined with immunoblotting:

  • Separate nuclei, cytosol, membrane, and chromatin-bound fractions

  • Perform Western blotting with At3g18150 antibody

  • Quantify relative distribution changes under stress conditions

  • Include compartment-specific markers for validation

• Immunofluorescence microscopy:

  • Fix plant tissues with 4% paraformaldehyde

  • Perform antigen retrieval if necessary (citrate buffer, pH 6.0)

  • Incubate with At3g18150 primary antibody (1:100-1:200)

  • Apply fluorophore-conjugated secondary antibody (1:200-1:500)

  • Co-stain with organelle markers

  • Analyze subcellular distribution before and after stress treatment

• Proximity ligation assay (PLA):

  • Use At3g18150 antibody together with antibodies against potential interactors

  • PLA signal indicates proximity (<40 nm) between proteins

  • Monitor interaction changes during stress progression

• Chromatin immunoprecipitation (ChIP):

  • If At3g18150 associates with chromatin during stress, perform ChIP followed by qPCR or sequencing

  • Map binding sites genome-wide and correlate with transcriptional changes

This multi-faceted approach will reveal how At3g18150 localization and interaction dynamics respond to environmental stresses, providing insights into its regulatory mechanisms.

What approaches can be used to study post-translational modifications of At3g18150 protein using antibodies?

Studying post-translational modifications (PTMs) of At3g18150 protein requires specialized antibody-based approaches:

• Modification-specific antibodies:

  • Generate antibodies against predicted phosphorylation, ubiquitination, or SUMOylation sites on At3g18150

  • Validate using synthetic phosphopeptides or in vitro modified recombinant protein

  • Apply in Western blots to detect modification status changes

• Two-dimensional gel electrophoresis with immunoblotting:

  • Separate proteins by isoelectric point and molecular weight

  • Transfer to membrane and probe with At3g18150 antibody

  • Shifts in position indicate modifications

  • Compare patterns before/after phosphatase treatment or stress conditions

• Immunoprecipitation coupled with mass spectrometry:

  • Immunoprecipitate At3g18150 using validated antibodies

  • Digest with trypsin and analyze by LC-MS/MS

  • Map modifications at amino acid resolution

  • Quantify modification stoichiometry under different conditions

• Phos-tag gel electrophoresis:

  • Incorporate Phos-tag molecules in SDS-PAGE to retard phosphorylated protein migration

  • Detect mobility shifts using At3g18150 antibody

  • Compare patterns with and without phosphatase treatment

These techniques enable researchers to chart the dynamic PTM landscape of At3g18150 and correlate modifications with protein function, stability, and localization.

How can At3g18150 antibodies be used in ChIP-seq experiments to identify DNA binding sites?

While At3g18150 is not annotated as a direct DNA-binding protein, it may associate with chromatin through interactions with transcription factors or chromatin modifiers. ChIP-seq using At3g18150 antibodies can reveal these associations:

• Chromatin preparation:

  • Crosslink Arabidopsis tissues with 1% formaldehyde for 10 minutes

  • Quench with glycine and isolate nuclei

  • Sonicate to generate 200-500 bp DNA fragments

  • Verify fragmentation by agarose gel electrophoresis

• ChIP optimization for At3g18150:

  • Test multiple antibody concentrations (2-10 μg per reaction)

  • Compare different washing stringencies

  • Include appropriate controls (IgG, input DNA, At3g18150 knockout)

  • Validate enrichment by qPCR at candidate regions before sequencing

• Data analysis pipeline:

  • Map reads to Arabidopsis genome (TAIR10 or latest)

  • Call peaks using MACS2 or similar algorithms

  • Perform motif discovery on enriched regions

  • Correlate binding sites with transcriptome data

  • Compare binding profiles under different conditions

• Validation experiments:

  • Confirm selected peaks by ChIP-qPCR

  • Perform sequential ChIP (Re-ChIP) to identify co-binding with known transcription factors

  • Test functional relevance using reporter assays

This approach can reveal if At3g18150 participates in transcriptional regulation through association with specific genomic regions, potentially connecting ubiquitin-mediated protein degradation with transcriptional control.

What are common issues when working with At3g18150 antibodies and how can they be resolved?

Several challenges may arise when working with antibodies against plant F-box proteins like At3g18150:

Always validate new antibody lots using positive controls and established protocols to ensure consistent performance across experiments.

How do post-translational modifications affect At3g18150 antibody recognition?

Post-translational modifications of At3g18150 can significantly impact antibody recognition:

• Epitope masking effects:

  • Phosphorylation near antibody epitopes may enhance or reduce binding

  • Ubiquitination can sterically block antibody access

  • Glycosylation may prevent antibody recognition completely

• Modification-dependent recognition patterns:

  • Some antibodies may preferentially recognize modified forms

  • Others may only bind unmodified epitopes

  • This results in differential detection of protein subpopulations

• Strategies for comprehensive detection:

  • Use multiple antibodies targeting different regions of At3g18150

  • Perform dephosphorylation assays to evaluate phosphorylation impact

  • Treat samples with deubiquitinating enzymes before analysis

  • Compare native versus denatured protein detection efficiency

• Characterization methods:

  • Treat recombinant At3g18150 with kinases, ubiquitin ligases or other modifying enzymes

  • Test antibody recognition of modified versus unmodified protein

  • Use synthetic peptides with defined modifications for epitope mapping

Understanding modification-dependent recognition patterns is crucial for accurate interpretation of At3g18150 detection results, especially when studying stress responses or developmental transitions where PTM profiles may change dramatically.

How can At3g18150 antibodies be used in multi-protein complex analysis?

Investigating At3g18150-containing protein complexes requires specialized approaches:

• Blue Native PAGE with immunoblotting:

  • Solubilize complexes using mild detergents (digitonin, DDM)

  • Separate intact complexes by BN-PAGE

  • Transfer to membrane and probe with At3g18150 antibody

  • Detect native complex size and composition

  • Excise bands for mass spectrometry identification

• Immunoprecipitation coupled with cross-linking:

  • Apply membrane-permeable crosslinkers (DSP, DTBP) to stabilize complexes

  • Perform IP with At3g18150 antibody

  • Analyze by SDS-PAGE followed by immunoblotting or mass spectrometry

  • Compare complex composition under different conditions

• Co-immunoprecipitation validation:

  • Perform reciprocal IPs with antibodies against identified interactors

  • Verify interactions by immunoblotting

  • Include appropriate controls (IgG, knockout lines)

• Size exclusion chromatography with immunodetection:

  • Fractionate plant extracts by size

  • Analyze fractions by immunoblotting for At3g18150

  • Identify co-eluting proteins by mass spectrometry

  • Map complex assembly/disassembly dynamics

This approach is particularly relevant for F-box proteins like At3g18150, which function in SCF complexes and may associate with different substrate recognition components depending on cellular context.

How do antibodies against At3g18150 compare with antibodies against related F-box proteins?

When working with antibodies against At3g18150 and related F-box proteins, researchers should consider:

• Epitope selection considerations:

  • F-box domain (N-terminal) is highly conserved and may lead to cross-reactivity

  • LRR regions offer greater specificity but may be less accessible in complex-bound protein

  • C-terminal regions often provide best specificity but may be subject to modifications

• Cross-reactivity profiles:

  • Test against recombinant proteins from most closely related family members

  • Perform immunoprecipitation followed by mass spectrometry to identify all bound proteins

  • Use knockout/knockdown lines of multiple family members for validation

• Functional domain accessibility:

  • F-box domain may be masked when protein is incorporated into SCF complex

  • LRR regions may change conformation upon substrate binding

  • Consider using multiple antibodies targeting different regions

• Application-specific performance:

  • Some antibodies may work well for Western blot but poorly for immunoprecipitation

  • Others may be ideal for immunofluorescence but inadequate for ChIP

  • Validate each antibody specifically for your application of interest

Understanding these comparative aspects helps in selecting the most appropriate antibody for specific experimental goals and in correctly interpreting results when studying F-box protein families.

What are emerging techniques for studying At3g18150 protein dynamics using antibody-based approaches?

Several cutting-edge techniques are expanding the toolkit for studying At3g18150 protein dynamics:

• Förster Resonance Energy Transfer (FRET) with antibody fragments:

  • Generate Fab fragments from At3g18150 antibodies

  • Label with donor fluorophores

  • Label potential interaction partner antibodies with acceptor fluorophores

  • Measure FRET in fixed or permeabilized cells

  • Track interaction dynamics with nanometer resolution

• Proximity-dependent biotin identification (BioID) coupled with antibody validation:

  • Express At3g18150-BioID fusion proteins in plants

  • Identify biotinylated proximal proteins by mass spectrometry

  • Validate interactions using At3g18150 antibodies in co-IP experiments

  • Create interaction network maps

• Super-resolution microscopy with antibody labeling:

  • Use At3g18150 antibodies with STORM or PALM super-resolution techniques

  • Achieve ~20 nm resolution of protein localization

  • Perform multi-color imaging to map protein neighborhood

  • Track reorganization during stress responses

• Single-molecule pull-down (SiMPull) assays:

  • Immobilize At3g18150 antibodies on microscope slides

  • Capture protein complexes from plant extracts

  • Visualize using total internal reflection fluorescence microscopy

  • Count individual complexes and determine stoichiometry

These emerging techniques offer unprecedented insights into At3g18150 protein dynamics, interaction networks, and functional relationships within the plant cell environment.

How can computational approaches enhance the design and application of At3g18150 antibodies?

Computational methods can significantly improve At3g18150 antibody development and experimental design:

• Epitope prediction and antibody design:

  • Use structural prediction algorithms to identify accessible epitopes on At3g18150

  • Analyze protein sequence conservation to select unique regions

  • Predict potential PTM sites that might interfere with antibody binding

  • Model antibody-antigen interactions to optimize affinity

• Network analysis for validation experiments:

  • Integrate transcriptomic and proteomic datasets to predict At3g18150 function

  • Identify key conditions for validation experiments

  • Predict potential interactors for co-immunoprecipitation validation

  • Map At3g18150 to stress response pathways

• Machine learning for image analysis:

  • Train algorithms to recognize specific immunostaining patterns

  • Automatically quantify protein colocalization from microscopy data

  • Detect subtle changes in localization under different conditions

  • Classify cellular responses based on At3g18150 dynamics

• Integrative multi-omics approaches:

  • Combine ChIP-seq, RNA-seq, and proteomics data

  • Correlate At3g18150 binding sites with gene expression changes

  • Predict functional outcomes of protein interactions

  • Generate testable hypotheses for antibody-based validation

By leveraging computational tools, researchers can design more specific antibodies, plan more informative experiments, and extract deeper biological insights from antibody-based studies of At3g18150 protein function in plant development and stress responses.