At3g10240 Antibody

What is the At3g10240 protein and why is it important in plant research?

At3g10240 is a putative F-box protein originally identified in Arabidopsis thaliana that has homologs in various plant species including Solanum lycopersicum (tomato) and Camelina sativa (false flax) . F-box proteins are part of the SCF ubiquitin-ligase complexes and play crucial roles in protein degradation pathways that regulate various cellular processes including hormone signaling, development, and stress responses in plants.

The protein has gained research interest because:

• It is conserved across multiple plant species, suggesting evolutionary importance

• F-box proteins often mediate specific protein-protein interactions in ubiquitination pathways

• Understanding its function may provide insights into plant developmental regulation

What types of antibodies against At3g10240 are typically used in research?

Researchers typically use polyclonal or monoclonal antibodies against At3g10240 depending on their experimental needs:

Custom-developed antibodies against plant proteins like At3g10240 are available from specialized research antibody providers that focus on plant model organisms like Arabidopsis .

How can I validate the specificity of an At3g10240 antibody?

Validation of At3g10240 antibodies should include multiple approaches:

• Western blot analysis using:

  • Wild-type plant tissue extracts (positive control)

  • At3g10240 knockout/knockdown mutant tissues (negative control)

  • Recombinant At3g10240 protein (positive control)

• Immunoprecipitation followed by mass spectrometry to confirm captured protein identity

• Competitive binding assays using recombinant At3g10240 protein to demonstrate specific binding

• Cross-reactivity testing against related F-box proteins to assess specificity

Remember that antibody validation is crucial for ensuring experimental reproducibility. A well-validated antibody should detect a band of the expected molecular weight (typically consistent with the predicted size of At3g10240) and show reduced or absent signal in knockout tissues.

What are the optimal conditions for using At3g10240 antibodies in Western blotting?

For optimal Western blot results with At3g10240 antibodies:

Sample preparation:

• Extract proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail

• For membrane-associated F-box proteins like At3g10240, consider using specialized extraction buffers that effectively solubilize membrane proteins

Blotting parameters:

• Transfer: Semi-dry transfer at 15V for 30 minutes or wet transfer at 30V overnight at 4°C

• Blocking: 5% non-fat dry milk in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature

• Primary antibody: Dilute At3g10240 antibody 1:1000 to 1:5000 in blocking buffer; incubate overnight at 4°C

• Secondary antibody: Anti-species IgG-HRP at 1:5000 to 1:10000 for 1 hour at room temperature

Optimization tips:

• Perform titration experiments to determine the optimal antibody dilution

• Consider using signal enhancers if the target protein is expressed at low levels

• For plant tissues with high phenolic compounds, add 2% PVPP to extraction buffer to prevent interference

How can I use At3g10240 antibodies for co-immunoprecipitation to study protein interactions?

Co-immunoprecipitation (Co-IP) is valuable for studying At3g10240 interactions with other SCF complex components or substrate proteins:

Protocol outline:

• Prepare plant tissue lysate in a gentle lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, protease inhibitors)

• Clear lysate by centrifugation (16,000 x g, 10 min, 4°C)

• Pre-clear with Protein A/G beads (1 hour, 4°C)

• Incubate pre-cleared lysate with At3g10240 antibody (2-5 μg) overnight at 4°C

• Add Protein A/G beads and incubate for 2-3 hours at 4°C

• Wash beads 4-5 times with wash buffer

• Elute complexes with SDS sample buffer or low pH glycine buffer

• Analyze by Western blot or mass spectrometry

Critical considerations:

• Cross-link the antibody to beads to prevent antibody co-elution

• Include appropriate controls (IgG control, input sample)

• For transient or weak interactions, consider crosslinking with DSP or formaldehyde

• Use gentle washing conditions to preserve protein-protein interactions

What are the best approaches for immunolocalization of At3g10240 in plant tissues?

Immunolocalization can reveal the subcellular distribution of At3g10240:

For confocal microscopy:

• Fix plant tissues in 4% paraformaldehyde in PBS for 30-60 minutes

• Permeabilize with 0.1-0.5% Triton X-100 for 15-30 minutes

• Block with 3% BSA in PBS for 1 hour

• Incubate with At3g10240 primary antibody (1:100-1:500) overnight at 4°C

• Wash 3x with PBS

• Incubate with fluorophore-conjugated secondary antibody for 1-2 hours

• Counterstain with DAPI for nuclei if desired

• Mount in anti-fade mounting medium

For transmission electron microscopy (TEM):

• Fix tissues in 4% paraformaldehyde/0.5% glutaraldehyde

• Embed in LR White resin

• Cut ultrathin sections (70-90 nm)

• Incubate with At3g10240 antibody followed by gold-conjugated secondary antibody

• Counterstain with uranyl acetate and lead citrate

Optimization tips:

• Always include controls (primary antibody omission, pre-immune serum)

• Validate specificity using knockout lines

• For challenging plant tissues, consider testing different fixatives and antigen retrieval methods

How can I use At3g10240 antibodies to investigate protein degradation pathways in plants?

Since At3g10240 is an F-box protein likely involved in protein degradation, antibodies against it can be powerful tools for studying ubiquitin-proteasome pathways:

Monitoring protein levels during degradation:

• Treat plant samples with proteasome inhibitors (MG132, 50 μM, 3-6 hours)

• Extract proteins at different time points

• Perform Western blotting with At3g10240 antibody

• Compare protein levels with and without inhibitor treatment

Detecting ubiquitinated targets:

• Perform At3g10240 immunoprecipitation

• Probe Western blots with anti-ubiquitin antibodies

• Identify ubiquitinated proteins by mass spectrometry

Cell-free degradation assays:

• Prepare plant cell extracts containing native ubiquitination machinery

• Add recombinant substrate proteins

• Monitor degradation over time by Western blotting

• Add At3g10240 antibodies to block specific degradation pathways

This approach allows researchers to determine if At3g10240 is directly involved in targeting specific substrates for degradation, providing insights into its functional role in plant cellular processes.

What strategies can I use to overcome cross-reactivity issues with At3g10240 antibodies in species with multiple F-box protein homologs?

F-box proteins often belong to large gene families with similar domains, presenting specificity challenges:

Epitope selection strategies:

• Target unique regions outside the conserved F-box domain

• Use peptide arrays to identify regions with minimal cross-reactivity

• Consider targeting post-translational modifications specific to At3g10240

Experimental approaches to mitigate cross-reactivity:

• Pre-absorption: Incubate antibody with recombinant homologous proteins to remove cross-reactive antibodies

• Knockout validation: Use genetic knockout lines to confirm signal specificity

• Serial immunodepletion: Sequentially deplete lysates of close homologs before detecting At3g10240

• Competitive ELISA: Use competing peptides to determine antibody specificity profiles

Data analysis considerations:

• Always include specificity controls in publications

• Clearly report which homologs were tested for cross-reactivity

• Consider computational analysis of epitope conservation across the F-box family

How can I quantitatively analyze At3g10240 protein expression across different plant tissues or developmental stages?

Quantitative analysis requires careful experimental design and appropriate controls:

ELISA-based quantification:

• Develop sandwich ELISA with capture and detection antibodies against different At3g10240 epitopes

• Generate a standard curve using recombinant At3g10240 protein

• Normalize protein expression to total protein content or housekeeping proteins

Quantitative Western blotting:

• Include recombinant protein standards of known concentration

• Use fluorescent secondary antibodies for wider linear range

• Analyze band intensities using software like ImageJ

• Normalize to loading controls (GAPDH, actin, tubulin)

Example quantification data for At3g10240 protein levels:

Statistical considerations:

• Perform at least 3 biological replicates

• Use appropriate statistical tests (ANOVA with post-hoc tests for multi-tissue comparison)

• Report confidence intervals and p-values

Why might I observe multiple bands when using At3g10240 antibodies in Western blots?

Multiple bands can result from various biological or technical factors:

Biological causes:

• Post-translational modifications (phosphorylation, ubiquitination)

• Alternative splice variants of At3g10240

• Protein degradation products

• Protein complexes not fully denatured

Technical causes:

• Cross-reactivity with related F-box proteins

• Non-specific binding

• Sample degradation during preparation

• Insufficient blocking

Verification strategies:

• Mass spectrometry: Excise bands and identify by MS

• Immunoprecipitation: Enrich the protein before Western blotting

• Genetic approach: Compare wild-type and knockout samples

• Peptide competition: Pre-incubate antibody with immunizing peptide

Example troubleshooting decision tree:

• Are bands consistently reproducible? → Yes: Likely biological; No: Technical issue

• Do bands disappear in knockout samples? → Yes: Specific signal; No: Potential cross-reactivity

• Do bands shift with treatments affecting post-translational modifications? → Yes: Modified forms of target protein

How can I develop and optimize a competitive ELISA for At3g10240 protein quantification?

Competitive ELISA can be particularly useful for plant proteins where matched antibody pairs are unavailable:

Development protocol:

• Coat plates with recombinant At3g10240 protein (1-5 μg/mL)

• Mix samples or standards with a fixed amount of At3g10240 antibody

• Add mixture to coated wells; free At3g10240 in samples competes with immobilized protein

• Detect bound antibody with enzyme-conjugated secondary antibody

• Develop with substrate and measure absorbance

Optimization parameters:

• Coating concentration (0.5-10 μg/mL)

• Antibody dilution (perform titration series)

• Sample dilution (create dilution series to ensure linearity)

• Incubation times and temperatures

Performance metrics to evaluate:

• Sensitivity (limit of detection)

• Specificity (cross-reactivity with homologs)

• Precision (intra- and inter-assay CV <15%)

• Accuracy (recovery of spiked samples 80-120%)

• Linearity (R² >0.95 for standard curve)

Similar competitive ELISA approaches have been demonstrated for other plant proteins and can be adapted for At3g10240 quantification in various plant tissues or experimental conditions .

How can I apply newer antibody-based technologies like AntiBinder to study At3g10240 interactions?

Recent advances in antibody technology can enhance At3g10240 research:

AntiBinder technology applications:
AntiBinder is a novel predictive model for antibody-antigen binding that integrates structural and sequence characteristics with a bidirectional cross-attention mechanism . For At3g10240 research, this could be applied to:

• Predict antibody-antigen binding:

  • Screen potential antibody candidates in silico before experimental production

  • Identify optimal epitopes that maximize specificity for At3g10240 over related F-box proteins

  • Optimize antibody design for specific applications (IP vs Western vs ELISA)

• Design bispecific antibodies:

  • Create antibodies that simultaneously target At3g10240 and interacting proteins

  • Develop detection systems for protein complexes containing At3g10240

• Cross-species applications:

  • Predict cross-reactivity with At3g10240 homologs in other plant species

  • Design pan-specific antibodies that recognize conserved epitopes across species

Implementation considerations:

• Provide sequence information of At3g10240 and potential antibody candidates to the AntiBinder algorithm

• Evaluate binding predictions based on bidirectional attention scores

• Validate computational predictions experimentally

The approach has demonstrated success in predicting antibody-antigen interactions, particularly for designing antibodies with reduced cross-reactivity to similar proteins .

What are the key considerations for using At3g10240 antibodies in multiplexed immunoassays with other plant proteins?

Multiplexed detection can provide insights into protein interaction networks:

Assay design principles:

• Antibody selection: Choose antibodies raised in different host species to allow distinct detection

• Cross-reactivity testing: Pre-test all antibodies for cross-reactivity with each other

• Signal separation: Use spectrally distinct fluorophores or unique reporter systems

• Sequential detection: Consider sequential rather than simultaneous detection if cross-reactivity occurs

Technical approaches:

• Multiplex Western blotting: Use antibodies from different species with spectrally distinct fluorescent secondaries

• Multiplex immunohistochemistry: Sequential labeling with careful stripping between rounds

• Flow cytometry: Multi-parameter analysis with differently labeled antibodies

• Proximity ligation assay (PLA): Detect At3g10240 interactions with other proteins in situ

Data analysis considerations:

• Account for potential signal bleed-through between channels

• Include single-stained controls for compensation calculations

• Use appropriate statistical methods for colocalization analysis

These multiplexed approaches can reveal relationships between At3g10240 and other proteins in the ubiquitin-proteasome pathway or identify novel interaction partners in different cellular compartments.

How should I document and report At3g10240 antibody validation data to ensure reproducibility?

Given the reproducibility crisis in biological research, proper documentation of antibody validation is critical:

Essential documentation:

• Antibody identifiers: Catalog number, lot number, RRID (Research Resource Identifier)

• Validation experiments performed: Western blot, IP, IF, knockout controls

• Experimental conditions: Dilutions, incubation times, buffers

• Positive and negative controls used

• Known cross-reactivity with other proteins

Recommended reporting format:

Best practices:

• Deposit detailed protocols in repositories like protocols.io

• Include representative validation images in publications or supplements

• Share validation data on antibody validation databases

• Consider using PLAbDab (the Patent and Literature Antibody Database) to find validated antibodies

What are the latest advancements in antibody-based detection that might enhance At3g10240 research?

Several emerging technologies show promise for plant protein research:

Single-cell antibody-based proteomics:

• Apply antibody-based detection at single-cell resolution

• Map At3g10240 expression across different cell types within plant tissues

• Combine with single-cell transcriptomics for multi-omics integration

Proximity-dependent biotinylation (BioID/TurboID):

• Fuse biotin ligase to At3g10240

• Identify proximal proteins in living cells

• Use anti-biotin antibodies to detect interaction networks

Nanobody technology:

• Develop camelid-derived single-domain antibodies against At3g10240

• Smaller size allows access to sterically hindered epitopes

• Can be expressed in vivo as intrabodies

Bispecific antibodies:

• Similar to those described in search result , bispecific antibodies targeting At3g10240 and potential interacting partners could be developed

• These would allow simultaneous binding to multiple targets

• Useful for detecting protein complexes in their native state

These technologies could significantly advance our understanding of At3g10240's role in plant biology by providing more sensitive, specific, and spatially resolved detection capabilities.

How can computational approaches enhance the design and application of At3g10240 antibodies?

Computational tools are increasingly important for antibody research:

Epitope prediction and antibody design:

• Use algorithms to identify unique epitopes in At3g10240 sequence

• Apply structural modeling to predict accessible regions

• Design antibodies with optimal binding properties

• Leverage AntiBinder-like approaches that utilize bidirectional attention mechanisms

Cross-reactivity prediction:

• Scan proteomes for similar epitopes to predict potential cross-reactivity

• Identify sequence regions unique to At3g10240 among F-box proteins

• Model binding affinity to related proteins

Data integration platforms:

• Combine antibody validation data with transcriptomics and proteomics

• Correlate antibody-based detection with other experimental methods

• Build integrative models of protein function and interaction

Machine learning applications:

• Train models on antibody binding data to improve epitope prediction

• Use large-scale antibody-antigen interaction datasets for training

• Develop models that predict antibody performance in different applications