At3g08810 Antibody

What is the At3g08810 protein and why is it studied in plant research?

At3g08810 encodes a F-box/kelch-repeat protein in Arabidopsis thaliana. F-box proteins are part of the SCF (Skp1-Cullin-F-box) complex that mediates protein ubiquitination and subsequent degradation via the 26S proteasome pathway. These proteins play crucial roles in various cellular processes including hormone signaling, cell cycle regulation, and developmental pathways.

The study of At3g08810 contributes to our understanding of protein degradation pathways and cellular regulation in plants. Researchers use antibodies against this protein to investigate its localization, expression patterns, and interactions with other proteins, which helps elucidate its function in plant development and stress responses.

What types of At3g08810 antibodies are available for plant research?

Based on current research resources, At3g08810 antibodies are available in the following formats:

These antibodies are typically generated using either synthetic peptides or recombinant proteins as immunogens. While peptide antibodies have been historically common, recombinant protein approaches have shown better success rates for plant proteins, with approximately 55% of antibodies showing high confidence signals in detection assays .

What standard applications are At3g08810 antibodies suitable for?

At3g08810 antibodies can be used for several standard applications in plant research:

• Western blotting (WB): For detecting the protein in plant tissue extracts and determining its expression levels

• Immunolocalization (IL): For visualizing the subcellular localization of the protein in plant tissues

• Immunoprecipitation (IP): For isolating the protein and its interacting partners

• ELISA: For quantitative measurement of protein levels

How should I design experiments to validate the specificity of At3g08810 antibody?

Validating antibody specificity is crucial for reliable results. A comprehensive validation approach for At3g08810 antibody should include:

• Genetic controls: Test the antibody on wild-type plants and at3g08810 mutant or knockout lines. A specific antibody should show a signal in wild-type plants but not in mutants lacking the target protein.

• Recombinant protein controls: Use purified recombinant At3g08810 protein as a positive control in Western blots to confirm the correct molecular weight detection.

• Preimmune serum controls: Compare results with preimmune serum to rule out non-specific binding from host animal antibodies.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide or protein before application. This should abolish specific signals.

• Cross-reactivity assessment: Test the antibody on closely related proteins to assess potential cross-reactivity, particularly with other F-box/kelch-repeat proteins.

Research has shown that affinity purification significantly improves detection rates for plant antibodies. In a study of 70 recombinant protein antibodies, affinity purification increased the success rate to 55%, with 22 antibodies suitable for immunocytochemistry .

What extraction methods are most effective for detecting At3g08810 protein in plant tissues?

For optimal extraction of At3g08810 protein from plant tissues:

• Buffer composition: Use a lysis buffer containing:

  • 50 mM Tris-HCl, pH 7.5

  • 150 mM NaCl

  • 1% Triton X-100 or NP-40

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

  • 1 mM PMSF

  • 5 mM DTT or β-mercaptoethanol

• Special considerations for F-box proteins:

  • Include 10-25 μM MG132 (proteasome inhibitor) to prevent degradation

  • Add deubiquitinating enzyme inhibitors (e.g., N-ethylmaleimide)

  • Consider phosphatase inhibitors if studying phosphorylation status

• Extraction procedure:

  • Grind plant tissue in liquid nitrogen to a fine powder

  • Add 3-5 volumes of extraction buffer per weight of tissue

  • Incubate with gentle agitation at 4°C for 30 minutes

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Collect supernatant for analysis

• Sample preparation for SDS-PAGE:

  • Mix with Laemmli buffer (with SDS and reducing agent)

  • Heat at 70°C for 10 minutes (avoid boiling which can cause aggregation of membrane-associated proteins)

  • Load 30-50 μg total protein per lane for Western blot analysis

How can I optimize immunolocalization protocols for At3g08810 antibody in plant tissues?

Optimizing immunolocalization for At3g08810 requires careful consideration of fixation, permeabilization, and detection methods:

• Tissue fixation:

  • Use 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours at room temperature

  • For better preservation of protein epitopes, try a milder fixative like 1-2% paraformaldehyde

  • Alternatively, use ethanol:acetic acid (3:1) for better penetration and antigen preservation

• Sectioning options:

  • Paraffin embedding: Better for morphological preservation

  • Cryosectioning: Often better for antigen preservation

  • Vibratome sectioning: Good for fresh tissue without embedding

• Permeabilization:

  • For cell wall permeabilization: 0.1-0.5% cellulase and 0.05% pectinase treatment

  • For membrane permeabilization: 0.1-0.3% Triton X-100 or 0.05-0.1% Tween-20

• Blocking and antibody incubation:

  • Block with 3-5% BSA or normal serum from the same species as secondary antibody

  • Use primary antibody at 1:100 to 1:1000 dilution (optimize through titration)

  • Incubate at 4°C overnight for best results

  • Include 0.1% Triton X-100 in antibody dilution buffer to reduce background

• Signal detection optimizations:

  • Test both fluorescent secondary antibodies and enzymatic detection systems

  • For low abundance proteins, consider tyramide signal amplification

  • Include appropriate controls (no primary antibody, preimmune serum)

Research on plant antibodies has shown that affinity-purified antibodies give significantly better results in immunolocalization studies compared to crude antisera .

What are the most common causes of false positives or negatives when using At3g08810 antibody?

Understanding potential sources of error is critical for accurate data interpretation:

Causes of false positives:

• Cross-reactivity with related proteins: The F-box/kelch-repeat protein family has conserved domains. At3g08810 antibodies may detect related proteins, especially if raised against conserved regions.

• Non-specific binding: Inadequate blocking or high antibody concentrations can lead to binding to unrelated proteins. Use 5% non-fat dry milk or BSA in TBST for blocking.

• Secondary antibody issues: Direct binding of secondary antibody to endogenous plant proteins, particularly those with Fc-binding properties. Include a secondary-only control.

• Endogenous peroxidases/phosphatases: These can cause background in enzymatic detection systems. Quench endogenous activities (e.g., 3% H₂O₂ for peroxidases).

Causes of false negatives:

• Epitope masking: Protein interactions, post-translational modifications, or fixation may mask the epitope. Try different extraction conditions or antigen retrieval methods.

• Protein degradation: F-box proteins are often unstable due to their role in degradation pathways. Include proteasome inhibitors (MG132) in extraction buffers.

• Low expression levels: At3g08810 may be expressed at low levels or in specific tissues/conditions. Enrich samples through immunoprecipitation or use more sensitive detection methods.

• Inadequate antigen retrieval: Formalin fixation can cross-link proteins, making epitopes inaccessible. Test different antigen retrieval methods (heat-induced, enzymatic, pH-based).

Research with plant antibodies has shown that even high-quality antibodies may require optimization for specific applications. In a study of 70 plant protein antibodies, only 55% showed high confidence signals after affinity purification .

How can I distinguish between specific and non-specific signals when using At3g08810 antibody?

Distinguishing specific from non-specific signals requires systematic controls and analysis:

• Essential controls:

  • Genetic controls: Compare signal between wild-type and at3g08810 knockout/knockdown plants

  • Peptide competition: Pre-incubate antibody with immunizing peptide/protein

  • Secondary antibody only: Assess background from secondary antibody

  • Pre-immune serum: Compare with the antibody signal

• Signal characteristics for specific binding:

  • Molecular weight: At3g08810 protein should appear at the expected molecular weight (~45-50 kDa, depending on post-translational modifications)

  • Subcellular localization: F-box proteins typically localize to nucleus and cytoplasm

  • Expression pattern: Should match known expression data from transcriptomics studies

  • Signal intensity: Should correlate with protein abundance in different tissues/conditions

• Quantitative analysis approaches:

  • Use densitometry software for Western blot quantification

  • Normalize to loading controls (e.g., actin, tubulin)

  • Compare signal-to-noise ratios between specific and control samples

  • Apply statistical tests to determine significance of observed differences

• Advanced validation:

  • Compare results from multiple antibodies targeting different epitopes

  • Correlate protein detection with mRNA expression data

  • Validate with tagged protein expression (e.g., GFP fusion)

How should I interpret contradictory results between Western blot and immunolocalization when using At3g08810 antibody?

Contradictory results between detection methods are common challenges in antibody-based research:

• Analysis of potential causes:

  Western Blot Result	Immunolocalization Result	Potential Explanation
  Positive	Negative	- Epitope masked by fixation - Protein denatured in Western but not in situ - Abundance below detection limit for immunolocalization
  Negative	Positive	- Cross-reactivity with related proteins in tissue - Protein lost during extraction - Aggregation during extraction preventing migration
  Multiple bands	Clean signal	- Degradation during extraction - Post-translational modifications - Splice variants detected on blot
  Single band	Diffuse/multiple locations	- Protein shuttles between compartments - Antibody cross-reactivity in tissue - Dynamic localization dependent on cell state

• Resolution strategies:

  • Modify fixation conditions for immunolocalization

  • Try different extraction methods for Western blotting

  • Use alternative detection methods (e.g., proximity ligation assay)

  • Validate with orthogonal approaches (e.g., mass spectrometry)

  • Test antibody on fractionated samples to confirm localization

• Interpretation framework:

  • Consider both results as complementary rather than contradictory

  • Assess which method has more appropriate controls

  • Evaluate which result aligns with known biology of F-box proteins

  • Consider if post-translational modifications explain the discrepancy

Research on plant antibodies has shown that different applications may require different optimization strategies. Some antibodies work well for Western blots but not for immunolocalization and vice versa .

How can At3g08810 antibody be used to study protein-protein interactions in the ubiquitin-proteasome pathway?

As an F-box protein, At3g08810 likely functions in the SCF complex for targeted protein degradation. The antibody can be leveraged for advanced interaction studies:

• Co-immunoprecipitation (Co-IP):

  • Use the At3g08810 antibody to pull down the protein complex

  • Identify interacting partners through Western blot or mass spectrometry

  • Protocol optimization:

    • Cross-link the antibody to protein A/G beads to prevent IgG contamination

    • Include proteasome inhibitors (MG132) to stabilize interactions

    • Try different salt concentrations to preserve weak interactions

    • Consider membrane-permeable crosslinkers before extraction

• Proximity-dependent labeling:

  • Create fusion proteins of At3g08810 with BioID or APEX2

  • Identify proximal proteins through streptavidin pulldown

  • Validate interactions with the At3g08810 antibody

• Immunofluorescence co-localization:

  • Double-label with At3g08810 antibody and antibodies against known SCF components

  • Quantify co-localization using Pearson's or Mander's coefficients

  • Perform FRET analysis to confirm direct interactions

• Chromatin immunoprecipitation (ChIP):

  • If At3g08810 functions in transcriptional regulation, use the antibody for ChIP

  • Identify DNA binding sites through sequencing or qPCR

Research on F-box proteins in plants has shown they often interact with specific substrates in a phosphorylation-dependent manner. Consider including phosphatase inhibitors when studying these interactions.

How can I use At3g08810 antibody to study protein degradation dynamics and stability?

The At3g08810 antibody can be valuable for investigating protein turnover and regulation:

• Cycloheximide chase assays:

  • Treat plants with cycloheximide to block protein synthesis

  • Collect samples at different time points

  • Use At3g08810 antibody in Western blots to track protein degradation

  • Quantify half-life through densitometry analysis

• Proteasome inhibitor studies:

  • Treat samples with MG132 or other proteasome inhibitors

  • Compare At3g08810 protein levels between treated and untreated samples

  • Identify stabilization of the protein and potential substrates

• Ubiquitination assays:

  • Immunoprecipitate with At3g08810 antibody

  • Probe with anti-ubiquitin antibodies to detect ubiquitinated forms

  • Alternative approach: Pull down with anti-ubiquitin and probe with At3g08810 antibody

• Stress-induced degradation:

  • Subject plants to various stresses (hormonal, abiotic, biotic)

  • Monitor changes in At3g08810 protein levels via Western blot

  • Correlate protein stability with stress responses and phenotypes

• Cell-free degradation assays:

  • Prepare plant cell extracts with active ubiquitin-proteasome system

  • Add recombinant At3g08810 protein

  • Monitor degradation kinetics in various conditions

The study of protein degradation dynamics is particularly relevant for F-box proteins like At3g08810, which are often themselves regulated by the ubiquitin-proteasome system through autoubiquitination.

What approaches can be used to study post-translational modifications of At3g08810 using the specific antibody?

Post-translational modifications (PTMs) of F-box proteins often regulate their function and stability:

• Phosphorylation analysis:

  • Immunoprecipitate At3g08810 using the antibody

  • Analyze by:

    • Western blot with phospho-specific antibodies

    • Phosphatase treatment to observe mobility shifts

    • Mass spectrometry to identify phosphorylation sites

  • Compare phosphorylation status under different conditions (e.g., hormone treatments)

• Ubiquitination detection:

  • Immunoprecipitate with At3g08810 antibody under denaturing conditions

  • Probe with anti-ubiquitin antibodies

  • Use mass spectrometry to map ubiquitination sites

  • Compare ubiquitination patterns in different tissues/conditions

• SUMOylation analysis:

  • Immunoprecipitate At3g08810

  • Probe with anti-SUMO antibodies

  • Identify SUMOylation sites by mass spectrometry

  • Study the effect of SUMOylation on protein stability and interactions

• 2D gel electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Use At3g08810 antibody to detect different isoforms

  • Cut spots for mass spectrometry analysis of PTMs

• Proximity ligation assay (PLA):

  • Use At3g08810 antibody along with antibodies against PTMs

  • Visualize and quantify modified forms in situ

Research has shown that F-box proteins are often regulated by phosphorylation, which can affect their stability, subcellular localization, and substrate recognition.

How can At3g08810 antibody be adapted for high-throughput or automated screening approaches?

Adapting the At3g08810 antibody for high-throughput applications requires specific optimization strategies:

• Antibody microarray development:

  • Immobilize At3g08810 antibody in microarray format

  • Screen for protein expression across multiple samples simultaneously

  • Optimize:

    • Antibody concentration for spotting (typically 0.5-1 mg/ml)

    • Surface chemistry (nitrocellulose, glass, hydrogel)

    • Detection method (fluorescence, chemiluminescence)

    • Blocking conditions to minimize background

• Automated immunohistochemistry:

  • Adapt protocol for automated staining platforms

  • Critical parameters:

    • Antibody dilution (typically higher than manual protocols)

    • Incubation time and temperature

    • Washing stringency

    • Detection system compatibility

• High-content screening:

  • Use At3g08810 antibody for immunofluorescence in multi-well formats

  • Combine with other markers for multiplexed analysis

  • Employ automated microscopy and image analysis

  • Measure parameters like:

    • Expression levels

    • Subcellular localization

    • Co-localization with other proteins

• Flow cytometry applications:

  • Optimize antibody for intracellular staining of protoplasts

  • Develop fixation and permeabilization protocols compatible with the antibody

  • Use fluorescent secondary antibodies for detection

  • Analyze distribution across cell populations

• Bead-based assays:

  • Couple At3g08810 antibody to magnetic beads

  • Develop multiplexed detection systems with other antibodies

  • Optimize bead concentration and sample volume ratios

For all high-throughput applications, verification of antibody specificity at scale is essential, as demonstrated by comprehensive antibody validation studies in plant systems .

How might At3g08810 antibody be used to investigate evolutionary conservation of F-box proteins across plant species?

The At3g08810 antibody can be leveraged for comparative studies across plant species:

• Cross-species reactivity testing:

  • Test the antibody on protein extracts from diverse plant species

  • Perform Western blots on:

    • Model plants (rice, maize, tomato, Brachypodium)

    • Evolutionarily diverse species (moss, ferns, gymnosperms)

    • Crop plants of economic importance

  • Correlate reactivity with sequence conservation

• Evolutionary proteomics approaches:

  • Immunoprecipitate homologous proteins from different species

  • Identify by mass spectrometry

  • Compare interacting partners across species

  • Map conservation of protein complexes

• Functional conservation analysis:

  • Use the antibody to study localization patterns across species

  • Compare expression patterns in response to stimuli

  • Correlate with phenotypic roles in different plant lineages

• Structural biology integration:

  • Use antibody-based purification for structural studies

  • Compare structural features of the protein across species

  • Relate structural conservation to functional conservation

This approach can provide insights into the evolution of the ubiquitin-proteasome system across plant lineages and reveal conserved regulatory mechanisms.

What emerging technologies could enhance the utility of At3g08810 antibody for plant research?

Several cutting-edge technologies can expand the research applications of At3g08810 antibody:

• Single-cell proteomics:

  • Adapt the antibody for single-cell Western blotting

  • Develop protocols for mass cytometry (CyTOF) with metal-conjugated antibodies

  • Explore spatial proteomics applications using imaging mass cytometry

• Super-resolution microscopy:

  • Optimize the antibody for techniques like STORM, PALM, or STED

  • Study nanoscale distribution and dynamics of At3g08810

  • Investigate co-localization with interacting partners at nanometer resolution

• Antibody engineering approaches:

  • Generate single-chain variable fragments (scFvs) from the antibody

  • Develop intrabodies for in vivo tracking of At3g08810

  • Create bispecific antibodies for simultaneous detection of At3g08810 and interacting proteins

• Live-cell applications:

  • Conjugate cell-penetrating peptides to the antibody

  • Develop protocols for intracellular delivery in intact plant cells

  • Monitor real-time dynamics of the protein

• Integration with CRISPR technologies:

  • Combine antibody detection with CRISPR-mediated tagging

  • Use the antibody to validate CRISPR-edited plants

  • Develop CUT&Tag protocols using the antibody for epigenomic profiling

These emerging technologies represent promising directions for expanding the research utility of At3g08810 antibody beyond conventional applications.

What are the recommended protocols for long-term storage and handling of At3g08810 antibody to maintain activity?

Proper storage and handling are critical for maintaining antibody functionality:

Storage recommendations:

• Stock solution storage:

  • Store at -20°C in small aliquots (20-50 μl) to avoid freeze-thaw cycles

  • Include 50% glycerol as a cryoprotectant

  • Add preservative (0.03% Proclin 300 or 0.02% sodium azide)

  • Maintain pH at 7.4 using phosphate-buffered saline

• Working dilution storage:

  • Store at 4°C for up to 2 weeks

  • Add protein carrier (0.1-1% BSA) to prevent adsorption to surfaces

  • Include 0.02% sodium azide to prevent microbial growth

• Stability monitoring:

  • Periodically test activity against positive control samples

  • Check for signs of aggregation (cloudiness) or contamination

  • Document activity over time at different storage conditions

Handling guidelines:

• Thawing procedure:

  • Thaw frozen aliquots at room temperature or 4°C (avoid heat)

  • Gently mix by finger-tapping (do not vortex)

  • Centrifuge briefly to collect all liquid at the bottom of the tube

• Contamination prevention:

  • Use sterile pipette tips and tubes

  • Avoid touching the inside of tube caps

  • Work in clean environment to prevent dust or microorganisms

• Transport conditions:

  • Ship with cold packs for short durations

  • Use dry ice for longer shipping times

  • Include temperature monitoring for valuable antibody shipments

Research has shown that the average shelf life of a properly stored antibody is 5-10 years, though activity should be verified periodically.

What positive and negative controls should be included when using At3g08810 antibody in different experimental setups?

Comprehensive controls are essential for robust antibody-based experiments:

Western blot controls:

Immunolocalization controls:

Immunoprecipitation controls: