At1g67130 Antibody

What is At1g67130 and what role does it play in Arabidopsis thaliana?

At1g67130 (UniProt: Q3ECH0) is a protein found in Arabidopsis thaliana (Mouse-ear cress), a model organism widely used in plant molecular biology research. The protein is implicated in plant developmental processes, potentially linked to gibberellin (GA) signaling pathways. GA-mediated signaling is critical for numerous plant developmental processes, including seed germination, stem elongation, and flowering. The At1g67130 protein may interact with DELLA proteins, which are known GA-signaling repressors that block GA-induced development . Understanding At1g67130's function can provide insights into fundamental plant growth regulatory mechanisms.

What are the primary applications of At1g67130 antibodies in plant research?

At1g67130 antibodies are valuable research tools primarily utilized in protein detection and characterization experiments. The main applications include:

• Western blotting (WB) for protein detection and quantification

• Enzyme-linked immunosorbent assay (ELISA) for sensitive protein quantification

• Immunoprecipitation (IP) for protein purification and interaction studies

• Chromatin immunoprecipitation (ChIP) for studying protein-DNA interactions

These applications enable researchers to investigate protein expression levels, post-translational modifications, protein-protein interactions, and protein localization within plant tissues and cells .

How should At1g67130 antibodies be properly stored and handled?

Proper storage and handling are essential for maintaining antibody functionality and experimental reproducibility. For At1g67130 antibodies:

• Store at -20°C or -80°C immediately upon receipt

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• Store in small aliquots to minimize freeze-thaw events

• When handling, keep the antibody on ice

• The antibody is typically provided in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

Improper storage can lead to antibody degradation, resulting in reduced sensitivity, increased background, and potentially misleading experimental results.

What protocol is recommended for Western blot analysis using At1g67130 antibodies?

When performing Western blot analysis with At1g67130 antibodies, researchers should follow this optimized protocol:

• Sample preparation:

  • Extract total protein from Arabidopsis tissues using appropriate buffer

  • Add protease inhibitors to prevent protein degradation

  • Quantify protein concentration using Bradford or BCA assay

• SDS-PAGE separation:

  • Load 20-50 μg of protein per lane

  • Use 10-12% polyacrylamide gels for optimal separation

• Transfer and blocking:

  • Transfer proteins to PVDF or nitrocellulose membrane

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute At1g67130 antibody (recommended starting dilution: 1:1000)

  • Incubate overnight at 4°C with gentle agitation

• Detection:

  • Use appropriate HRP-conjugated secondary antibody

  • Develop using ECL substrate

  • Visualize using X-ray film or digital imaging system

• Controls:

  • Include positive control (Arabidopsis wild-type extract)

  • Include negative control (extract from At1g67130 knockout line if available)

  • Use loading control antibody (e.g., against actin or tubulin)

The expected molecular weight for At1g67130 protein should be verified based on sequence prediction and previous literature, as antibody specificity validation is critical for accurate results.

How can researchers validate the specificity of At1g67130 antibodies?

Antibody specificity validation is critical, especially considering the documented issues with non-specificity in some commercial antibodies. For At1g67130 antibodies, implement these validation steps:

• Genetic validation:

  • Compare Western blot results between wild-type and At1g67130 knockout/knockdown plants

  • The specific band should be absent or significantly reduced in the knockout/knockdown samples

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide

  • This should abolish specific binding in Western blot or immunostaining

• Recombinant protein control:

  • Use purified recombinant At1g67130 protein as a positive control

  • Verify expected molecular weight and antibody recognition

• Multiple antibody comparison:

  • Use antibodies raised against different epitopes of At1g67130

  • Consistent results with different antibodies increase confidence in specificity

• Orthogonal techniques:

  • Complement antibody-based approaches with non-antibody methods (e.g., mass spectrometry)

  • Correlate protein detection with mRNA expression data

These validation approaches are particularly important given that studies have shown many commercial antibodies can exhibit cross-reactivity and non-specific binding .

What are the optimal parameters for immunoprecipitation using At1g67130 antibodies?

For successful immunoprecipitation of At1g67130 and its interaction partners:

• Lysate preparation:

  • Use fresh Arabidopsis tissue (preferably young seedlings)

  • Grind tissue in liquid nitrogen to fine powder

  • Extract proteins in non-denaturing buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitors)

  • Clear lysate by centrifugation at 14,000g for 15 minutes at 4°C

• Pre-clearing:

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add 2-5 μg of At1g67130 antibody per 500 μg of protein lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add 30 μl Protein A/G beads

  • Incubate for 2-3 hours at 4°C

• Washing and elution:

  • Wash beads 4-5 times with cold IP buffer

  • Elute proteins with SDS sample buffer at 95°C for 5 minutes

• Analysis:

  • Analyze by Western blot or mass spectrometry

  • Include proteasome inhibitor MG132 (10-50 μM) in some experiments to preserve ubiquitinated forms of the protein

For detecting protein-protein interactions, particularly those with DELLA proteins or other components of gibberellin signaling pathways, co-immunoprecipitation experiments have proven effective in similar studies .

How can At1g67130 antibodies be used to investigate light and gibberellin signaling crosstalk?

At1g67130 antibodies can elucidate the functional relationships between light and gibberellin signaling pathways using these approaches:

• ChIP-seq analysis:

  • Use At1g67130 antibodies for chromatin immunoprecipitation followed by sequencing

  • Identify genome-wide binding sites under different light conditions

  • Compare binding patterns with and without GA treatment

  • Map binding sites to known light and GA-responsive gene promoters

• Protein complex analysis:

  • Combine At1g67130 immunoprecipitation with mass spectrometry

  • Identify interaction partners under different light × hormone treatment combinations

  • Verify interactions using reciprocal co-immunoprecipitation

  • Analyze how complex formation changes with environmental stimuli

• Phosphorylation state analysis:

  • Immunoprecipitate At1g67130 under different signaling conditions

  • Analyze post-translational modifications by mass spectrometry

  • Determine how phosphorylation states change with light quality or GA treatment

  • Create phospho-specific antibodies for key regulatory sites

• Protein stability assays:

  • Use At1g67130 antibodies to measure protein half-life

  • Monitor protein levels after cyclohexamide treatment

  • Compare stability with and without GA treatment

  • Assess ubiquitination patterns under different light conditions

This approach is supported by previous research showing how GA and light signaling pathways coordinate developmental processes in Arabidopsis, potentially through protein-protein interactions and post-translational modifications of key signaling components .

What methodological approaches can be used to study At1g67130 interactions with DELLA proteins?

DELLA proteins are key repressors in GA signaling pathways, and their interaction with At1g67130 can be studied using these approaches:

• Bimolecular Fluorescence Complementation (BiFC):

  • Create fusion constructs of At1g67130 and DELLA proteins with split YFP fragments

  • Express in tobacco leaves via Agrobacterium-mediated transformation

  • Visualize interactions through fluorescence microscopy

  • Include appropriate controls (non-interacting proteins)

• In vitro pull-down assays:

  • Express and purify recombinant At1g67130 and DELLA proteins from bacteria

  • Perform pull-down assays with and without GA

  • Analyze interaction using Western blot with At1g67130 antibodies

  • Include DNA probes to test DNA-dependence of interactions

• Co-immunoprecipitation with endogenous proteins:

  • Immunoprecipitate using At1g67130 antibodies

  • Detect DELLA proteins (RGA, GAI, etc.) in the precipitate

  • Compare interactions with and without GA treatment

  • Include proteasome inhibitors (MG132) to preserve ubiquitinated forms

• Yeast two-hybrid analysis:

  • Create fusion constructs with DNA-binding and activation domains

  • Test direct interactions between At1g67130 and DELLA proteins

  • Use deletion mutants to map interaction domains

  • Verify interactions with different methods

These approaches have been successfully used to study interactions between DELLA proteins and other transcription factors in Arabidopsis, revealing how GA signaling modulates protein interactions to regulate plant development .

How can researchers use At1g67130 antibodies to study protein dynamics during plant development?

To investigate the dynamic changes in At1g67130 protein levels, modifications, and interactions during plant development:

• Temporal expression profiling:

  • Collect Arabidopsis tissues at different developmental stages

  • Perform Western blot analysis using At1g67130 antibodies

  • Quantify protein levels relative to appropriate loading controls

  • Correlate protein levels with developmental transitions

• Spatial localization studies:

  • Perform immunohistochemistry on tissue sections

  • Use fluorescent secondary antibodies for detection

  • Analyze protein localization in different cell types

  • Compare localization patterns at different developmental stages

• Chromatin association dynamics:

  • Conduct ChIP experiments at key developmental transitions

  • Map changes in genomic binding sites during development

  • Correlate binding with changes in target gene expression

  • Identify cofactors that modulate chromatin association

• Protein modification tracking:

  • Immunoprecipitate At1g67130 from tissues at different stages

  • Analyze post-translational modifications by mass spectrometry

  • Track changes in phosphorylation, ubiquitination, or other modifications

  • Correlate modifications with protein function and stability

These approaches can reveal how At1g67130 function changes throughout the plant life cycle, particularly in response to environmental cues that trigger developmental transitions through light and hormone signaling pathways.

How can researchers troubleshoot non-specific binding issues with At1g67130 antibodies?

Non-specific binding is a common challenge with antibodies. For At1g67130 antibodies, implement these troubleshooting strategies:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Increase blocking time or concentration

  • Add 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Adjust antibody concentration:

  • Perform a dilution series (1:500 to 1:5000) to identify optimal concentration

  • Reduce primary antibody concentration if background is high

  • Extend incubation time when using more dilute antibody

• Modify washing protocol:

  • Increase number of washes (5-6 times)

  • Extend wash duration (10-15 minutes each)

  • Add higher salt concentration (up to 500 mM NaCl) to reduce ionic interactions

• Pre-adsorb antibody:

  • Incubate antibody with proteins from non-target species

  • For plant studies, pre-adsorb with proteins from unrelated plant species

  • Remove non-specific antibodies by centrifugation

• Validate with knockout controls:

  • Compare staining patterns between wild-type and At1g67130 knockout plants

  • Any bands/signals present in knockout samples represent non-specific binding

Remember that many commercial antibodies show cross-reactivity with unintended targets. Research has documented cases where antibodies detected identical bands in wild-type and knockout tissues, highlighting the importance of proper controls and validation .

What strategies can help resolve contradictory results when using At1g67130 antibodies from different sources?

When facing contradictory results with different At1g67130 antibodies:

• Comprehensive antibody validation:

  • Validate each antibody using knockout/knockdown controls

  • Perform peptide competition assays for each antibody

  • Identify the specific epitopes recognized by each antibody

• Multi-method confirmation:

  • Validate findings using orthogonal techniques (e.g., mass spectrometry)

  • Correlate protein data with mRNA expression analysis

  • Use genetic approaches (overexpression, CRISPR knockouts) to confirm findings

• Systematic comparison:

  • Test all antibodies under identical experimental conditions

  • Document differences in immunization antigens, host species, and purification methods

  • Consider differences in antibody format (polyclonal vs. monoclonal)

• Epitope accessibility analysis:

  • Different antibodies may detect different conformational states

  • Some epitopes may be masked by protein-protein interactions

  • Post-translational modifications may affect antibody recognition

• Independent replication:

  • Have different lab members repeat experiments

  • Collaborate with other labs to validate findings

  • Document all experimental conditions in detail

Systematic evaluation is particularly important given that studies have found that commercially available antibodies to related proteins (e.g., AT1 receptor) produced different immunostaining patterns unrelated to the presence or absence of the target protein .

How should researchers report and interpret At1g67130 antibody data in publications?

To ensure scientific rigor when reporting At1g67130 antibody data:

These practices align with increasing recognition in the scientific community that antibody validation is essential for reproducible research, particularly given documented cases of non-specific antibody binding in the literature .

What are the best approaches for designing experiments to study At1g67130 ubiquitination patterns?

To effectively investigate At1g67130 ubiquitination:

• Ubiquitination detection protocol:

  • Treat plants with proteasome inhibitors (e.g., MG132, 50 μM for 4-6 hours)

  • Include deubiquitinase inhibitors in lysis buffer (N-ethylmaleimide, 10 mM)

  • Immunoprecipitate At1g67130 using validated antibodies

  • Detect ubiquitinated forms by Western blot with anti-ubiquitin antibodies

• Differentiate ubiquitination types:

  • Use antibodies specific for K48-linked chains (associated with degradation)

  • Use antibodies specific for K63-linked chains (associated with signaling)

  • Compare ubiquitination patterns with and without GA treatment

  • Analyze how light conditions affect ubiquitination patterns

• Identify ubiquitination sites:

  • Perform mass spectrometry on immunoprecipitated At1g67130

  • Focus on lysine residues with ubiquitin remnants

  • Create lysine-to-arginine mutants to test functional significance

  • Compare ubiquitination sites under different conditions

• Study ubiquitination machinery:

  • Identify E3 ligases that interact with At1g67130

  • Use co-immunoprecipitation with At1g67130 antibodies

  • Test candidate E3 ligases based on GA signaling components

  • Analyze At1g67130 stability in E3 ligase mutants

Previous research has shown that GA treatment enhances the detection of high-molecular-weight DELLA protein species that react with anti-ubiquitin antibodies, suggesting a similar approach may be effective for At1g67130 .

How can At1g67130 antibodies be applied in genome-wide studies of protein-DNA interactions?

For genome-wide mapping of At1g67130 binding sites:

• ChIP-seq optimization:

  • Cross-link Arabidopsis seedlings with 1% formaldehyde for 10 minutes

  • Sonicate chromatin to 200-300 bp fragments

  • Immunoprecipitate using At1g67130 antibodies

  • Include appropriate controls (IgG, input DNA)

  • Prepare libraries for high-throughput sequencing

• Experimental design considerations:

  • Compare binding profiles under different light conditions

  • Analyze GA-treated versus untreated samples

  • Include time-course experiments for developmental transitions

  • Compare wild-type to related mutants (e.g., DELLA mutants)

• Data analysis approach:

  • Use peak-calling algorithms optimized for plant ChIP-seq

  • Perform motif discovery analysis on bound regions

  • Correlate binding sites with gene expression data

  • Integrate with published datasets on light and GA responses

• Validation of binding sites:

  • Confirm selected binding sites by ChIP-qPCR

  • Test functional significance with reporter gene assays

  • Perform EMSA with recombinant At1g67130 protein

  • Use DNA affinity purification followed by Western blotting

These approaches have been successfully used to study DNA-binding proteins involved in light and hormone signaling in Arabidopsis, revealing genomic targets and regulatory mechanisms .

What considerations are important when using At1g67130 antibodies in different plant species?

When applying At1g67130 antibodies across plant species:

• Cross-reactivity assessment:

  • Perform sequence alignment of At1g67130 orthologs in target species

  • Focus on the epitope region recognized by the antibody

  • Test antibody reactivity in the new species by Western blot

  • Include positive (Arabidopsis) and negative controls

• Optimization for each species:

  • Adjust protein extraction protocols for species-specific tissues

  • Modify blocking conditions to reduce background

  • Titrate antibody concentration for optimal signal-to-noise ratio

  • Validate with genetic resources when available

• Conservative interpretation:

  • Be cautious when interpreting cross-species results

  • Confirm findings with species-specific antibodies when possible

  • Support antibody data with genomic or transcriptomic evidence

  • Consider raising new antibodies against species-specific orthologs

• Functional validation across species:

  • Compare protein function between Arabidopsis and target species

  • Assess conservation of protein interactions

  • Test complementation of mutants across species

  • Analyze conservation of expression patterns and regulation

These considerations are particularly important given documented issues with antibody specificity. Even within the same species, commercially available antibodies can show variable specificity and different staining patterns .