At1g23770 Antibody

What is the At1g23770 gene and what protein does it encode?

At1g23770 encodes a putative F-box protein in Arabidopsis thaliana that likely functions in protein-protein interactions and ubiquitin-mediated protein degradation pathways . F-box proteins typically serve as substrate recognition components within SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes that target specific proteins for degradation, playing crucial roles in various cellular processes including hormone signaling and development.

What are the common applications for At1g23770 antibodies in plant research?

At1g23770 antibodies can be utilized for:

• Western blotting to detect protein expression levels

• Immunohistochemistry to visualize protein localization in plant tissues

• Immunoprecipitation to isolate protein complexes

• Chromatin immunoprecipitation (if the protein has DNA-binding properties)

• Enzyme-linked immunosorbent assays (ELISA)

Similar plant protein antibodies have been successfully used for immunocytochemistry and in situ immunolocalization to determine subcellular localization patterns .

How are antibodies against plant proteins like At1g23770 typically generated?

Antibodies against plant proteins can be generated using two main approaches:

Synthetic peptide approach:

• Selection of unique, antigenic peptide sequences (typically 10-15 amino acids)

• Conjugation to carrier proteins like KLH

• Immunization of host animals (rabbits, sheep, mice)

Recombinant protein approach:

• Expression of protein fragments in bacterial systems

• Purification of the recombinant protein

• Immunization of host animals

As noted in search result , the recombinant protein approach often yields better results for plant proteins, with success rates around 55% compared to very low success rates with peptide approaches .

What types of At1g23770 antibodies are available for research?

Though specific At1g23770 antibodies aren't directly referenced in the search results, plant protein antibodies generally fall into two categories:

Polyclonal antibodies:

• Generated from multiple B cell lineages

• Recognize multiple epitopes on the target protein

• Generally higher sensitivity but may have lower specificity

• Typically produced in rabbits, sheep, or goats

Monoclonal antibodies:

• Generated from a single B cell clone

• Recognize a single epitope

• Higher specificity but may have lower sensitivity

• Typically produced using hybridoma technology in mice

What strategies should be employed for validating At1g23770 antibody specificity?

Comprehensive validation should include:

• Western blot analysis using:

  • Wild-type Arabidopsis tissue

  • At1g23770 knockout/mutant lines

  • Tissues with known expression levels

• Testing for cross-reactivity with:

  • Recombinant At1g23770 protein

  • Related F-box proteins

• Peptide competition assays:

  • Pre-incubation of antibody with antigenic peptide

  • Should eliminate specific signal

• Heterologous expression systems:

  • Detection in systems expressing tagged At1g23770

Several antibodies in the plant research community have been validated using knockout/mutant backgrounds, which provides strong evidence for specificity .

How do post-translational modifications affect At1g23770 antibody binding?

Post-translational modifications (PTMs) can significantly impact antibody recognition:

Researchers should consider generating antibodies against different regions of At1g23770 to account for potential modifications, especially since F-box proteins often undergo regulatory modifications themselves.

What are the challenges in generating specific antibodies for At1g23770?

Key challenges include:

• Sequence homology with related F-box proteins: F-box proteins share conserved domains, making unique epitope selection critical to prevent cross-reactivity.

• Epitope prediction limitations: As noted in search result , "prediction methods identify individual stretches of amino acids (continuous epitopes), whereas epitopes are very often discontinuous, involving distant subsequences brought together by the protein's tertiary structures."

• Expression levels: At1g23770 may be expressed at low levels or in specific tissues/conditions, complicating antibody generation and validation.

• Protein conformation: Recombinant proteins may not fold correctly, resulting in antibodies that fail to recognize the native protein structure .

• Antibody purification requirements: Research indicates that affinity purification significantly improves detection rates for plant antibodies (from very low to approximately 55%) .

How can immunohistochemistry with At1g23770 antibodies be optimized for plant tissues?

Optimization strategies include:

• Fixation protocol selection:

  • Aldehyde-based fixatives (paraformaldehyde, glutaraldehyde)

  • Duration and temperature of fixation critical for epitope preservation

• Antigen retrieval methods:

  • Heat-induced epitope retrieval

  • Enzymatic digestion for cell wall components

• Signal amplification techniques:

  • Tyramide signal amplification

  • Polymer-based detection systems

• Background reduction:

  • Pre-adsorption with plant tissue extracts

  • Optimization of blocking reagents (BSA, milk, normal serum)

• Specificity controls:

  • Pre-immune serum controls

  • Knockout/mutant tissue controls

  • Peptide competition controls

Recent research with plant antibodies shows immunolocalization success rates improve dramatically with properly affinity-purified antibodies .

What is the recommended Western blot protocol for At1g23770 antibodies?

Based on successful protocols for plant protein antibodies:

• Sample preparation:

  • Extract proteins using buffer containing:

    • 50-200 mM Tris-HCl (pH 7.5-8.0)

    • 0.1-0.2% Triton X-100

    • 0.1 mM EDTA

    • Protease inhibitors

  • Heat samples at 70-80°C for 5-10 minutes

• Gel electrophoresis:

  • Load 10-15 μg total protein

  • Use 10-12% SDS-PAGE gels

• Transfer:

  • PVDF or nitrocellulose membranes (0.2 μm pore size)

  • Semi-dry or wet transfer (75V for 45 min to 1 hour)

• Blocking:

  • 3-5% non-fat milk or BSA in TBS-T

  • 1 hour at room temperature or overnight at 4°C

• Antibody incubation:

  • Primary antibody: 1:1000 dilution in blocking solution

  • Overnight at 4°C with gentle agitation

  • Secondary antibody: 1:5000-1:20000 HRP-conjugated

• Detection:

  • ECL chemiluminescence

  • Exposure times from 30 seconds to 2 minutes

This protocol is adapted from successfully tested plant antibodies as described in search results .

How should samples be prepared for immunoprecipitation with At1g23770 antibodies?

Optimal immunoprecipitation protocol:

• Tissue harvesting:

  • Flash-freeze tissue in liquid nitrogen

  • Grind to fine powder using mortar and pestle

• Protein extraction:

  • Extract in non-denaturing buffer:

    • 50 mM Tris-HCl pH 7.5

    • 150 mM NaCl

    • 1% NP-40 or 0.5% Triton X-100

    • 1 mM EDTA

    • Protease inhibitor cocktail

    • Phosphatase inhibitors (if phosphorylation is relevant)

• Pre-clearing:

  • Incubate lysate with Protein A/G beads

  • 1 hour at 4°C with rotation

• Immunoprecipitation:

  • Add 2-5 μg antibody per 500 μg protein

  • Incubate overnight at 4°C with rotation

  • Add Protein A/G beads

  • Incubate 2-4 hours at 4°C

• Washing:

  • 4-5 washes with IP buffer

  • Final wash with 50 mM Tris-HCl pH 7.5

• Elution:

  • SDS sample buffer for Western blot analysis

  • Gentle elution buffer for functional assays

Based on successful immunoprecipitation methods mentioned in search result , this protocol should be effective for At1g23770.

What controls are essential when performing immunofluorescence with At1g23770 antibodies?

Essential controls include:

• Negative controls:

  • Secondary antibody only (no primary)

  • Pre-immune serum at the same dilution as immune serum

  • At1g23770 knockout/mutant tissue

• Specificity controls:

  • Peptide competition (pre-absorption with immunizing peptide)

  • Serial dilution of primary antibody

• Positive controls:

  • Known subcellular marker antibodies

  • GFP-tagged At1g23770 (if available)

• Technical controls:

  • Autofluorescence control (unstained sample)

  • Counterstaining (DAPI for nuclei, etc.)

These controls help distinguish true signals from artifacts, particularly important since subcellular localization information can provide crucial insights into F-box protein function.

How can cross-reactivity with related F-box proteins be assessed and minimized?

To assess and minimize cross-reactivity:

• In silico analysis:

  • Sequence alignment of At1g23770 with related F-box proteins

  • Identification of unique regions for antibody generation

  • Bioinformatic cut-off: <40% sequence similarity for antigenic regions

• Experimental assessment:

  • Western blot against recombinant related F-box proteins

  • Testing in tissues with known expression patterns of related proteins

  • Testing in mutants of related F-box proteins

• Improvement strategies:

  • Affinity purification against the specific protein/peptide

  • Pre-adsorption with related proteins

  • Epitope selection away from the conserved F-box domain

• Validation approach:

  • Expression comparison with transcript data

  • Correlation with phenotypic changes in knockout/overexpression lines

According to search result , careful bioinformatic analysis to identify unique antigenic regions significantly improves antibody specificity.

What troubleshooting approaches are recommended for weak or non-specific signals with At1g23770 antibodies?

Systematic troubleshooting approaches:

According to search result , "affinity purification with the purified recombinant protein resulted in significant improvement in detection rate: 38 (55%) antibodies could detect a signal with high confidence either by in situ immunolocalization (22 out of 38) or Westerns (20 out of 32 tested) or both."

How can At1g23770 antibodies be utilized in protein-protein interaction studies?

Advanced approaches include:

• Co-immunoprecipitation:

  • Precipitation with At1g23770 antibody

  • Western blot for potential interacting partners

  • Mass spectrometry for unbiased interaction discovery

• Proximity-dependent labeling:

  • BioID or APEX2 fusion with At1g23770

  • Antibody detection of biotinylated proteins

• Förster resonance energy transfer (FRET):

  • Fluorophore-conjugated At1g23770 antibody

  • Detection of energy transfer to antibodies against candidate interactors

• Fluorescence co-localization:

  • At1g23770 antibody plus antibodies to potential partners

  • Quantitative co-localization analysis

These methods are particularly relevant for F-box proteins like At1g23770, which function through protein-protein interactions within SCF complexes.

Can antibodies against At1g23770 be used in plant species beyond Arabidopsis thaliana?

Cross-species applicability depends on:

• Sequence conservation:

  • Analysis of At1g23770 homologs in target species

  • Epitope conservation assessment

• Validation approaches:

  • Western blot with tissue from multiple species

  • Testing recombinant proteins from related species

• Cross-reactivity potential:

  • Higher for polyclonal antibodies against conserved regions

  • Lower for monoclonal antibodies against specific epitopes

Many plant antibodies show cross-reactivity with related species. For example, the antibody against Arabidopsis GOX shows reactivity with proteins from Pisum sativum, Oryza sativa, and several other plant species .

What are the advantages and limitations of monoclonal versus polyclonal antibodies for At1g23770 research?

As seen in search result , monoclonal antibodies can provide broader neutralizing activity against diverse targets, which might be relevant for detecting At1g23770 under different conditions or conformations.

How do recent advances in antibody engineering apply to plant protein research with At1g23770?

Emerging antibody technologies relevant to At1g23770 research:

• Recombinant antibody fragments:

  • Single-chain variable fragments (scFvs)

  • Fab fragments

  • Nanobodies (VHH antibodies)

• CRISPR-based knock-in strategies:

  • Endogenous tagging of At1g23770 for antibody-free detection

  • Generation of epitope-tagged lines for validated antibody targets

• Bispecific antibodies:

  • Simultaneous detection of At1g23770 and interacting partners

  • Approach described in search result for enhanced specificity

• Synthetic antibody libraries:

  • Phage display selection of high-affinity binders

  • Avoids animal immunization

These approaches may overcome traditional limitations in generating antibodies against challenging plant proteins like At1g23770.

What considerations are important for quantitative analysis of At1g23770 using antibody-based techniques?

For accurate quantification:

• Standardization approaches:

  • Inclusion of recombinant protein standards

  • Use of housekeeping protein controls

  • Standard curve generation for each experiment

• Signal quantification methods:

  • Densitometry for Western blots

  • Fluorescence intensity measurement for immunofluorescence

  • ELISA for precise concentration determination

• Statistical considerations:

  • Biological and technical replicates (minimum n=3)

  • Appropriate statistical tests for data analysis

  • Normalization strategies

• Validation of quantitative data:

  • Correlation with mRNA levels

  • Comparison with alternative protein quantification methods

  • Verification in multiple tissue types/conditions

• Dynamic range limitations:

  • Antibody saturation effects

  • Linear range determination for quantification