At1g60710 Antibody

What is the AT1G60710 gene and what protein does it encode?

AT1G60710 is a protein-coding gene in Arabidopsis thaliana that encodes ATB2 protein . The gene is located on chromosome 1 and serves as a model for studying plant protein expression systems. When conducting research with antibodies targeting this protein, it's essential to understand that ATB2 is part of the complex regulatory network in Arabidopsis and may be involved in plant stress responses, although its precise function requires further characterization through antibody-based techniques such as immunoblotting and immunolocalization.

What are the key considerations for validating an AT1G60710 antibody?

Antibody validation requires a comprehensive approach to ensure specificity, selectivity, and reproducibility in your experimental context. For AT1G60710 antibodies, the following validation steps are recommended:

• Western blot analysis - Verify a single band at the expected molecular weight for ATB2 protein

• Negative controls - Use non-expressing tissues or knockout lines of Arabidopsis as controls

• Positive controls - Include samples with known expression or overexpression of AT1G60710

• Cross-reactivity assessment - Test against closely related proteins to confirm specificity

• Application-specific validation - Validate independently for each application (WB, IHC, IF, etc.)
  This "fit for purpose" validation approach ensures your antibody is appropriate for your specific experimental design and biological context .

What sample preparation methods work best for plant protein immunodetection using AT1G60710 antibodies?

Sample preparation significantly impacts antibody performance in plant tissues. For optimal results with AT1G60710 antibodies:

• Protein extraction: Use extraction buffer containing components like:

  • 25 mM Tris-HCl, pH 7.6

  • 15 mM MgCl₂

  • 150 mM NaCl

  • Protease inhibitors (e.g., 1 µM E64, PMSF)

  • 0.1% Nonidet P-40

• Sample processing:

  • Determine protein concentration using Bradford assay

  • Denature in Laemmli buffer

  • Run on 4-15% TGX gels at 300V for 20 minutes

  • Transfer to 0.2-µm PVDF membranes

• Fixation considerations: Time to fixation, fixation period, and fixative type will affect tissue antigenicity and epitope availability

How should I design experiments to distinguish between specific and non-specific binding of AT1G60710 antibodies?

Designing robust experiments requires multiple controls to distinguish true signal from background:

• Mixed population experiments: Create mixtures with defined proportions of AT1G60710-expressing and non-expressing tissues to establish a standard curve

• Control hierarchy:

  • No primary antibody control

  • Isotype control

  • Non-expressing tissue control (most critical)

  • Knockdown/knockout plant lines (gold standard)

• Orthogonal validation: Compare antibody results with alternative methods of protein detection:

  • mRNA expression analysis

  • Mass spectrometry identification

  • Fluorescent protein tagging

• Sample preparation comparison: Test multiple fixation and extraction methods, as demonstrated in Fig. 1 of reference , where different sample preparations affected antibody performance differently between positive and negative populations.

What is the recommended immunoblot protocol for detecting AT1G60710 protein in plant samples?

For reliable immunoblot detection of AT1G60710:

• Protein extraction and quantification:

  • Extract total protein using buffer with protease inhibitors

  • Determine concentration (30 μg protein recommended for loading)

• SDS-PAGE and transfer:

  • Separate proteins on 12% acrylamide gels

  • Transfer electrophoretically to nitrocellulose membrane (45 μm)

  • Block with 10% (w/v) fat-free milk

• Antibody incubation:

  • Primary antibody: Anti-AT1G60710 (dilution 1:3000-1:5000 based on antibody specifications)

  • Incubate overnight at 4°C

  • Secondary antibody: Anti-rabbit IgG-HRP (1:10,000)

  • Incubate 60 min at room temperature

• Detection:

  • Enhanced chemiluminescence (ECL)

  • Include loading control (anti-actin, Agrisera AS13 2640, 1:3000)

How can I use antibodies to study protein-protein interactions involving AT1G60710?

To investigate protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use magnetic Protein A/G beads with crosslinked antibodies

  • Prepare stromal extract (6 mg protein/mL)

  • Supplement with RNAsin (1 unit/μL)

  • Pre-clear by centrifugation (10 min at 18,000g)

  • Analyze precipitated complexes by mass spectrometry

• Tandem affinity purification (TAP):

  • Generate tagged AT1G60710 constructs (e.g., protein G/streptavidin-binding peptide tag)

  • Express in Arabidopsis cell cultures

  • Isolate protein complexes under native conditions

  • Identify interaction partners using MS analysis

• Proximity labeling approaches:

  • BioID or TurboID fusions to AT1G60710

  • Expression in plant cells followed by streptavidin pulldown

  • Mass spectrometry identification of proximal proteins

What are the common issues when using AT1G60710 antibodies in Western blots and how can they be resolved?

Common Western blot issues and solutions:

How can I address non-specific binding of AT1G60710 antibodies in immunolocalization experiments?

To minimize non-specific binding in immunolocalization:

• Pre-adsorption controls:

  • Incubate antibody with purified antigen before use

  • Compare staining pattern with and without pre-adsorption

• Blocking optimization:

  • Test different blocking solutions (BSA, normal serum, milk)

  • For plant tissues, try PBS-buffered 5% BSA at 20°C for 50 minutes

• Sample preparation considerations:

  • Different fixatives affect epitope accessibility

  • Compare multiple sample preparation methods to determine optimal conditions

  • Test paraformaldehyde vs. methanol fixation

• Antibody dilution optimization:

  • Perform titration experiments (typical dilution range: 1:500-1:1000)

  • Include proper negative controls at each dilution

What controls should I include when validating AT1G60710 antibodies for flow cytometry experiments?

For flow cytometry validation:

• Essential controls:

  • Unstained cells (autofluorescence control)

  • Isotype control (non-specific binding)

  • Secondary antibody only (background control)

  • Known negative cell population

  • Known positive cell population

• Mixed population experiment:

  • Create defined mixtures of positive and negative cells

  • Compare known percent composition to experimentally determined percent positivity

  • Plot correlation to validate quantitative accuracy

• Sample preparation comparison:

  • Test multiple sample preparation protocols

  • Compare results between preparations using the same antibody clone

  • Evaluate how preparation affects both positive and negative populations

How can I use AT1G60710 antibodies to study protein post-translational modifications?

For studying post-translational modifications (PTMs):

• Modification-specific antibodies:

  • Generate antibodies against predicted modification sites

  • Validate using synthetic peptides with and without modifications

• Two-dimensional analysis:

  • First dimension: Separate based on PTM status using specific antibodies

  • Second dimension: Verify identity with AT1G60710 antibodies

• Mass spectrometry validation:

  • Immunoprecipitate AT1G60710 using validated antibodies

  • Analyze by MS to identify modification sites

  • Example from research: AT1G60710-related JAZ12 was found to be modified with di-Gly at Lys-169, indicating ubiquitination or similar modification

• Comparative studies:

  • Compare modification status under different stress conditions

  • Use phospho-specific antibodies when applicable, following validation principles

What are the considerations for using AT1G60710 antibodies in plant stress response studies?

When studying stress responses:

• Experimental design considerations:

  • Include appropriate time course sampling

  • Compare multiple stress types (cold, drought, heat, oxidative, etc.)

  • Use proper unstressed controls

• Target validation approach:

  • Consider using the TARGET (Transient Assay Reporting Genome-wide Effects of Transcription factors) system

  • Combine with antibody detection for protein-level verification

• Specific controls for stress studies:

  • Stress-responsive known proteins as positive controls

  • Constitutively expressed proteins (e.g., actin) as loading controls

  • Verify antibody specificity under stress conditions, as protein modifications may change

• Multi-omics integration:

  • Correlate antibody-based protein detection with transcriptomic data

  • Consider protein localization changes during stress response

How can I develop and validate custom antibodies against specific domains or variants of AT1G60710?

For developing custom antibodies:

• Epitope selection strategies:

  • Analyze protein domains for unique, accessible regions

  • Consider hydrophilicity, surface probability, and antigenicity

  • Use bioinformatics tools to identify domain-specific epitopes

• Production approaches:

  • Recombinant protein fragments (100-150 amino acids)

  • Synthetic peptides conjugated to carrier proteins

  • For AT1G60710, consider using fragments with N-terminal hexahistidine tags expressed in E. coli

• Comprehensive validation process:

  • Test against recombinant protein

  • Verify with knockout/knockdown lines

  • Perform epitope mapping

  • Conduct specificity testing against related proteins

  • Validate independently for each application (WB, IHC, IF)

• Documentation requirements:

  • Record complete information about immunogen

  • Document all validation experiments

  • Follow standardized reporting guidelines as recommended for antibody research

How should I interpret contradictory results between antibody-based techniques and other methods when studying AT1G60710?

When facing contradictory results:

• Systematic troubleshooting approach:

  • Verify antibody specificity with additional controls

  • Consider technical limitations of each method

  • Example: In studying psbA RNA, researchers found apparent contradictions between different methods that were resolved by comparing multiple antibody approaches

• Methodological reconciliation:

  • Consider that protein and mRNA levels may not correlate

  • Proteins may be differentially localized or modified

  • Antibodies may detect specific conformations or modifications

• Cross-validation strategies:

  • Use orthogonal techniques (e.g., MS vs. antibody detection)

  • Apply genetic approaches (CRISPR/Cas9 editing)

  • Test multiple antibodies against different epitopes of AT1G60710

What standards should I follow when reporting AT1G60710 antibody usage in publications?

Follow these reporting standards:

• Essential antibody information:

  • Complete antibody identification (source, catalog number, lot number, RRID)

  • Host species and clonality

  • Immunogen details and epitope information

• Validation documentation:

  • Describe all validation experiments performed

  • Include appropriate positive and negative controls

  • Present evidence of specificity for each application used

• Protocol details:

  • Provide complete experimental conditions

  • Specify dilutions, incubation times, and temperatures

  • Describe blocking conditions and washing steps

• Proper statistical analysis:

  • Include replication information

  • Provide quantification methods

  • Report variability between experiments

• Data availability:

  • Consider depositing raw data in appropriate repositories

  • Make validation data available upon request