At1g59675 Antibody

What is the At1g59675 protein and why is it significant for plant research?

At1g59675 encodes a putative F-box protein in Arabidopsis thaliana. It belongs to one of the largest superfamilies of regulatory proteins in plants, with Arabidopsis containing at least 568 F-box protein genes . F-box proteins are integral components of SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes that regulate protein degradation via the ubiquitin-proteasome pathway. This protein may be involved in drought stress adaptation and other developmental processes, making it a target of interest for plant biologists studying stress responses and developmental regulation .

What types of At1g59675 antibodies are currently available for research?

Currently, polyclonal antibodies against At1g59675 are commercially available. These include rabbit anti-Arabidopsis thaliana At1g59675 polyclonal antibodies suitable for various applications including ELISA and Western blotting . Most available antibodies are developed to recognize the full-length protein rather than specific domains or post-translationally modified versions .

What is the typical domain structure of At1g59675 that antibodies might recognize?

At1g59675 contains an approximately 40-50 amino acid long F-box domain at its N-terminal region. Additionally, it has C-terminal domains that may include:

• F-box associated domain type 1 (FBA1) spanning from 127 to 365 amino acids

• F-box associated domain type 3 (FBA3) spanning from 233 to 357 amino acids

• Immunoglobulin-like fold that may be involved in protein-protein interactions

This domain structure should be considered when selecting antibodies for specific research purposes, as some may preferentially recognize certain domains.

How should I validate the specificity of At1g59675 antibodies in my experimental system?

To validate antibody specificity for At1g59675, implement a multi-step approach:

• Western blotting: Use tissue from wild-type plants and At1g59675 knockout/knockdown mutants to confirm antibody specificity. The expected molecular weight of At1g59675 is approximately 34.34 kDa .

• Co-localization experiments: If investigating subcellular localization, perform immunofluorescence experiments with markers for cellular compartments (nucleus and membrane) since At1g59675 has been shown to localize to both .

• Co-immunoprecipitation: Verify interaction partners by pulling down At1g59675 and confirming interactors like ASK1, Cullin1, or ADA2b through mass spectrometry or immunoblotting .

• Blocking peptide: Use the specific peptide used to generate the antibody to confirm signal specificity. Pre-incubation with this peptide should abolish the signal in immunodetection experiments.

What are the optimal methods for detecting At1g59675 protein expression in different plant tissues?

For comprehensive detection of At1g59675 across plant tissues:

• Tissue preparation: Extract proteins using buffer containing protease inhibitors to prevent degradation. Consider that At1g59675 has higher expression in roots compared to other organs , so sample preparation should be optimized accordingly.

• Western blotting protocol:

  • Use 10-12% SDS-PAGE gels

  • Transfer proteins to PVDF or nitrocellulose membrane

  • Block with 5% non-fat milk or BSA

  • Incubate with At1g59675 antibody (typically at 1:500-1:2000 dilution)

  • Use appropriate secondary antibody (anti-rabbit IgG for polyclonal antibodies)

  • Develop using ECL or fluorescent detection systems

• Immunohistochemistry:

  • Fix tissue samples in 4% paraformaldehyde

  • Embed in paraffin or prepare frozen sections

  • Perform antigen retrieval if necessary

  • Block endogenous peroxidase activity

  • Incubate with At1g59675 antibody followed by appropriate detection system

• Flow cytometry (for cell-specific analysis):

  • Prepare plant protoplasts

  • Fix and permeabilize cells

  • Stain with At1g59675 antibody and fluorophore-conjugated secondary antibody

  • Analyze using flow cytometer

How can I optimize immunoprecipitation protocols using At1g59675 antibodies?

For successful immunoprecipitation of At1g59675:

• Sample preparation:

  • Extract plant proteins under non-denaturing conditions

  • Use buffer containing 150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl (pH 7.5), and protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation status

• Antibody immobilization:

  • Couple At1g59675 antibody to protein A/G magnetic beads or agarose

  • Crosslink antibody to beads using dimethyl pimelimidate (DMP) to prevent co-elution

• Immunoprecipitation:

  • Incubate lysate with antibody-coupled beads (4-16 hours at 4°C)

  • Wash extensively with buffer containing decreasing salt concentrations

  • Elute bound proteins using pH shift or competitive elution with the immunizing peptide

• Verification:

  • Analyze immunoprecipitates by Western blotting for At1g59675

  • Use mass spectrometry to identify interaction partners

  • Confirm functional interactions through additional biochemical assays

How can I use At1g59675 antibodies to study SCF complex formation and function?

To investigate SCF complex formation involving At1g59675:

• Co-immunoprecipitation studies:

  • Immunoprecipitate At1g59675 and probe for SCF components (ASK1, Cullin1)

  • Perform reverse co-IP with antibodies against known SCF components

  • Use crosslinking agents to stabilize transient interactions

• In vitro reconstitution:

  • Express and purify recombinant At1g59675, ASK1, and Cullin1

  • Perform pull-down assays to assess complex formation

  • Analyze by size-exclusion chromatography to determine complex stoichiometry

• Ubiquitination assays:

  • Set up in vitro ubiquitination reactions with reconstituted SCF complexes

  • Use potential substrate proteins (like transcriptional co-activator ADA2b)

  • Detect ubiquitinated products by Western blotting

• Functional analysis:

  • Compare ubiquitination activity between wild-type and mutant forms of At1g59675

  • Assess effects of environmental stressors (drought, ABA treatment) on complex activity

  • Correlate with phenotypic observations from At1g59675 mutant plants

How can At1g59675 antibodies help investigate drought stress responses in plants?

For studying At1g59675's role in drought stress adaptation:

• Expression analysis:

  • Quantify At1g59675 protein levels in plants under normal and drought conditions

  • Compare expression patterns with transcript analyses showing altered expression in response to mannitol and ABA treatment

• Target protein analysis:

  • Immunoprecipitate At1g59675 from control and drought-stressed plants

  • Identify differential interaction partners using mass spectrometry

  • Focus on proteins involved in drought response pathways

• Cellular localization changes:

  • Perform immunofluorescence or subcellular fractionation followed by immunoblotting

  • Determine if drought stress alters At1g59675 localization between nucleus and membrane

  • Correlate with functional changes in protein interactions or degradation

• Functional validation:

  • Compare protein levels of known drought response factors (RD29A, RD22, ABI3) in wild-type versus At1g59675 mutant plants

  • Assess correlation between At1g59675 activity and accumulation of stress markers like H₂O₂ and malondialdehyde (MDA)

What methodological approaches can be used to investigate At1g59675 interactions with transcriptional regulators?

To study interactions between At1g59675 and transcriptional regulators:

• Yeast two-hybrid validation:

  • Clone At1g59675 into bait vectors (such as pGBKT7BD)

  • Screen against transcriptional regulators like ADA2b in prey vectors (such as pGADT7)

  • Verify interactions on selective media (SD/-Leu/-Trp/-His/-Ade/X-α-gal/Aureobasidin)

• In vivo proximity labeling:

  • Generate BioID or TurboID fusions with At1g59675

  • Express in Arabidopsis protoplasts or stable transgenic lines

  • Identify proximal proteins through streptavidin pull-down and mass spectrometry

• ChIP-Western analysis:

  • Perform chromatin immunoprecipitation with antibodies against transcription factors

  • Probe immunoprecipitates for At1g59675 to determine association with chromatin complexes

  • Alternatively, immunoprecipitate At1g59675 and probe for DNA enrichment

• Functional transcriptional assays:

  • Develop reporter gene assays with promoters regulated by At1g59675's interaction partners

  • Compare reporter activity in wild-type, At1g59675 overexpression, and knockout backgrounds

  • Assess how these interactions affect expression of drought-responsive genes

What are the common challenges in Western blotting with At1g59675 antibodies and how can they be overcome?

Common Western blotting challenges and solutions:

• Weak or no signal:

  • Increase antibody concentration (try 1:500 instead of 1:1000)

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

  • Use more sensitive detection systems (enhanced chemiluminescence)

  • Enrich samples through immunoprecipitation before Western blotting

  • Check protein extraction method - F-box proteins can be labile during extraction

• Multiple bands or high background:

  • Increase blocking time and concentration (5% milk for 2 hours)

  • Add 0.1-0.5% Tween-20 in wash buffers

  • Perform more wash steps (5× 10 minutes)

  • Use antigen-affinity purified antibodies as they typically have higher specificity

  • Consider using knockout/knockdown controls to identify specific bands

• Inconsistent results between experiments:

  • Prepare fresh protein samples (F-box proteins may be unstable)

  • Standardize protein quantification methods

  • Include loading controls specific for cellular compartments where At1g59675 localizes

  • Consider that expression levels may vary with growth conditions and tissue types

• Detection of protein degradation products:

  • Add proteasome inhibitors (MG132) to extraction buffers

  • Include deubiquitinase inhibitors (PR-619) if studying ubiquitination

  • Perform extraction at 4°C with complete protease inhibitor cocktails

How can I distinguish between At1g59675 and other F-box proteins that might cross-react with the antibody?

To ensure specificity and distinguish from other F-box proteins:

• Sequence alignment analysis:

  • Before selecting an antibody, analyze sequence similarity between At1g59675 and other F-box proteins

  • Choose antibodies raised against unique regions with low homology to other F-box proteins

  • The C-terminal region tends to be more diverse among F-box proteins

• Validation using genetic materials:

  • Use knockout/knockdown lines of At1g59675 as negative controls

  • Check for signal disappearance in these lines to confirm specificity

  • Test antibody against recombinant proteins of closely related F-box family members

• Immunodepletion experiments:

  • Pre-incubate antibody with purified At1g59675 protein

  • Use depleted antibody preparation in parallel experiments

  • True signals should disappear after depletion

• Mass spectrometry validation:

  • Immunoprecipitate proteins recognized by the antibody

  • Perform mass spectrometry analysis to confirm identity

  • Check for peptides unique to At1g59675 versus other F-box proteins

What considerations are important when designing experiments to study post-translational modifications of At1g59675?

For investigating post-translational modifications (PTMs):

• Sample preparation:

  • Use phosphatase inhibitors (sodium orthovanadate, sodium fluoride) for phosphorylation studies

  • Include deubiquitinase inhibitors for ubiquitination studies

  • Add HDAC inhibitors for acetylation studies

  • Consider native extraction conditions to preserve labile modifications

• Modification-specific detection:

  • Use modification-specific antibodies (anti-phospho, anti-ubiquitin) in combination with At1g59675 antibodies

  • Perform sequential immunoprecipitation (first with At1g59675 antibody, then with modification-specific antibody)

  • Consider using Multiple Reaction Monitoring (MRM) mass spectrometry for targeted PTM detection

• Functional relevance assessment:

  • Compare PTM patterns between normal and stress conditions (drought, salt, heat)

  • Generate site-specific mutants (S/T→A for phosphorylation, K→R for ubiquitination)

  • Correlate PTM status with protein function, localization, and interactions

• PTM site mapping:

  • Immunoprecipitate At1g59675 using the antibody

  • Perform mass spectrometry analysis with PTM-enrichment strategies

  • Validate identified sites through site-directed mutagenesis and functional assays

How can At1g59675 antibodies be used in high-throughput screening approaches to identify novel interaction partners?

For high-throughput interaction partner identification:

• Immunoprecipitation coupled with mass spectrometry:

  • Perform large-scale immunoprecipitation from different tissues and conditions

  • Analyze by LC-MS/MS to identify co-precipitating proteins

  • Use label-free quantification or SILAC to compare interactomes between conditions

  • Filter against datasets from unrelated antibodies to remove common contaminants

• Protein microarray applications:

  • Probe protein microarrays containing Arabidopsis proteome with recombinant At1g59675

  • Alternatively, use the antibody to detect At1g59675 on microarrays probed with potential interactors

  • Compare binding profiles between control and stress conditions

• Yeast two-hybrid screens:

  • Use At1g59675 as bait against normalized cDNA libraries

  • Focus on tissue-specific libraries (root tissue shows highest expression)

  • Validate hits through secondary assays using the antibody (co-IP, pull-down)

• Proximity-dependent labeling:

  • Generate BioID or TurboID fusions with At1g59675

  • Express in Arabidopsis and identify biotinylated proteins

  • Compare with control TurboID fusions to identify specific interactions

  • Validate key interactions using traditional co-IP with At1g59675 antibodies

What methodological approaches can integrate At1g59675 antibody data with genomic and transcriptomic datasets?

For multi-omics integration strategies:

• ChIP-seq applications:

  • Use At1g59675 antibodies for chromatin immunoprecipitation

  • Sequence associated DNA to identify potential genomic binding sites

  • Correlate with transcriptomic data to identify genes potentially regulated by At1g59675 complexes

  • Focus on drought-responsive genes like RD29A, RD22, and ABI3

• Proteogenomic approaches:

  • Compare At1g59675 protein levels (by quantitative immunoblotting) with transcript levels

  • Identify conditions where post-transcriptional regulation occurs

  • Correlate with data from interactome studies to build regulatory networks

• Phenomic correlation:

  • Quantify At1g59675 protein levels across ecotypes with varying drought tolerance

  • Correlate with phenotypic data and known genetic variants

  • Use machine learning to identify patterns associated with stress responses

• Systems biology modeling:

  • Incorporate At1g59675 protein level data into existing regulatory network models

  • Test hypotheses regarding its role in F-box protein networks

  • Predict effects of perturbations through computational simulations and validate experimentally

How can quantitative analysis of At1g59675 be performed in different subcellular compartments?

For quantitative subcellular distribution analysis:

• Subcellular fractionation with immunoblotting:

  • Separate nuclear, membrane, cytosolic, and other fractions

  • Quantify At1g59675 in each fraction by immunoblotting

  • Use compartment-specific markers to validate fractionation purity

  • Compare distributions between normal and stress conditions

• Immunofluorescence quantification:

  • Perform immunofluorescence labeling with At1g59675 antibodies

  • Co-stain with compartment markers (nuclear, membrane, etc.)

  • Quantify colocalization using fluorescence microscopy and image analysis

  • Apply deconvolution algorithms for increased spatial resolution

• Live-cell imaging approaches:

  • Generate fluorescent protein fusions that can be detected with anti-GFP antibodies

  • Validate localization pattern matches endogenous protein using At1g59675 antibodies

  • Track dynamic changes in localization during stress responses

  • Perform FRAP (Fluorescence Recovery After Photobleaching) to assess mobility

• Mass spectrometry-based spatial proteomics:

  • Combine subcellular fractionation with stable isotope labeling

  • Immunoprecipitate At1g59675 from each fraction

  • Quantify relative abundances by mass spectrometry

  • Identify compartment-specific interaction partners

What are the most advanced methods for studying the dynamics of At1g59675-containing protein complexes?

For studying dynamic protein complexes:

• Size-exclusion chromatography with immunodetection:

  • Separate native protein complexes by size

  • Analyze fractions by immunoblotting with At1g59675 antibodies

  • Identify changes in complex size under different conditions

  • Reanalyze key fractions by mass spectrometry to identify complex components

• Blue native PAGE with antibody detection:

  • Separate intact protein complexes under native conditions

  • Transfer to membrane and probe with At1g59675 antibodies

  • Excise gel spots for mass spectrometry analysis of complex components

  • Compare complex patterns between wild-type and mutant plants

• Single-molecule tracking:

  • Use fluorescently labeled antibody fragments (Fab) for live-cell imaging

  • Track individual molecules/complexes using super-resolution microscopy

  • Analyze diffusion coefficients and binding kinetics

  • Correlate with cellular responses to environmental stimuli

• Hydrogen-deuterium exchange mass spectrometry:

  • Immunoprecipitate At1g59675 complexes using the antibody

  • Perform H/D exchange to probe structural dynamics

  • Analyze by mass spectrometry to identify regions with altered solvent accessibility

  • Compare exchange patterns between functional states or conditions