At3g07870 Antibody

What is At3g07870 and why is it studied in Arabidopsis research?

At3g07870 is a protein encoded by the At3g07870 gene in Arabidopsis thaliana (Mouse-ear cress), which serves as a model organism in plant biology. The protein has a Uniprot identification number of Q9SFC7 and is primarily studied in the context of fundamental plant biology research. Research involving At3g07870 contributes to our understanding of plant cellular functions and responses to environmental stimuli. The antibody against this protein enables researchers to detect, localize, and quantify the At3g07870 protein in various experimental settings .

What are the validated applications for At3g07870 antibody?

The At3g07870 antibody has been validated for use in Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) applications. These techniques allow researchers to detect and quantify the target protein in complex biological samples. In Western Blotting, the antibody enables the identification of the target protein based on molecular weight after separation by gel electrophoresis. For ELISA, the antibody facilitates quantitative detection of the target protein in solution. Both applications have been specifically tested to ensure proper identification of the antigen .

What is the recommended storage protocol for At3g07870 antibody?

Upon receipt, the At3g07870 antibody should be stored at either -20°C or -80°C to maintain its activity and specificity. Researchers should avoid repeated freeze-thaw cycles as these can degrade antibody performance. The antibody is supplied in liquid form with a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative. This formulation helps maintain antibody stability during long-term storage. For short-term usage (within a week), aliquots can be kept at 4°C, but long-term storage requires freezing to prevent antibody degradation .

How should I design positive and negative controls for experiments using At3g07870 antibody?

When designing controls for At3g07870 antibody experiments, include:

Positive controls:

• Wild-type Arabidopsis thaliana tissue samples known to express At3g07870

• Recombinant At3g07870 protein (ideally the same used as immunogen)

• Transfected cell lines overexpressing At3g07870

Negative controls:

• Arabidopsis knockout/knockdown lines lacking At3g07870 expression

• Non-plant tissue samples or distantly related plant species

• Primary antibody omission controls

• Blocking peptide competition assays to confirm specificity

These controls help validate antibody specificity and provide reference points for interpreting experimental results, particularly in complex systems where multiple proteins may share structural similarities .

How can I optimize Western blot protocols specifically for At3g07870 detection?

Optimizing Western blot protocols for At3g07870 detection requires attention to several parameters:

• Sample preparation: Extract proteins from Arabidopsis tissues using a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

• Gel selection: Use 10-12% polyacrylamide gels for optimal resolution

• Transfer conditions:

  • 100V for 60 minutes in standard Towbin buffer

  • Consider semi-dry transfer systems for more efficient protein transfer

• Blocking optimization:

  • 5% non-fat dry milk in TBST (preferred)

  • Alternatively, 3% BSA in TBST if background is high

• Antibody dilution: Begin with 1:1000 dilution and optimize as needed

• Detection system:

  • Enhanced chemiluminescence for standard detection

  • Fluorescent secondary antibodies for quantitative analysis

When troubleshooting, focus on membrane washing steps (use at least 3×10 minutes with TBST) and consider overnight primary antibody incubation at 4°C to improve sensitivity and specificity .

What approaches should I use to validate At3g07870 antibody specificity in my experimental system?

Validating antibody specificity for At3g07870 requires a multi-faceted approach:

• Genetic validation:

  • Test antibody against wild-type and At3g07870 knockout/knockdown Arabidopsis lines

  • Analyze tissue-specific expression patterns matching known mRNA profiles

• Biochemical validation:

  • Perform pre-adsorption tests using the immunizing peptide

  • Conduct immunoprecipitation followed by mass spectrometry

  • Run parallel Western blots with different antibody clones (if available)

• Molecular validation:

  • Use siRNA-mediated knockdown to confirm reduced signal

  • Test against overexpression systems showing increased signal

  • Perform epitope mapping to confirm binding specificity

• Cross-reactivity testing:

  • Test against related Arabidopsis proteins

  • Check cross-reactivity with homologous proteins from other plant species

This comprehensive validation ensures experimental results can be interpreted with confidence and minimizes the risk of false positives or negatives in your research system .

How can I disentangle potential binding modes of At3g07870 antibody when working with multiple epitopes?

When working with At3g07870 antibody in complex systems with multiple potential epitopes, consider the following biophysics-informed approach:

• Computational epitope mapping:

  • Use bioinformatics tools to predict potential epitopes on At3g07870

  • Compare with closely related proteins to identify unique regions

• Experimental binding mode analysis:

  • Employ peptide arrays covering overlapping segments of At3g07870

  • Use alanine scanning mutagenesis to identify critical binding residues

  • Consider hydrogen-deuterium exchange mass spectrometry to identify antibody binding regions

• Competitive binding assays:

  • Use fragments of the target protein to compete for antibody binding

  • Analyze binding kinetics with surface plasmon resonance (SPR)

  • Implement Bio-Layer Interferometry to measure association/dissociation rates

• Cross-specificity testing:

  • Test against synthetic peptides representing potential cross-reactive epitopes

  • Create a specificity profile using closely related protein variants

This methodical approach allows researchers to characterize distinct binding modes and can help optimize experimental conditions for improved specificity and reduced cross-reactivity .

What are the most common causes of background signal when using At3g07870 antibody in immunoassays?

Background signal issues when using At3g07870 antibody typically arise from:

• Non-specific antibody binding:

  • Insufficient blocking (increase blocking reagent concentration to 5-7%)

  • Inadequate washing (extend wash steps to 4×15 minutes)

  • Secondary antibody cross-reactivity (test secondary alone without primary)

• Sample-related issues:

  • Excessive protein loading (reduce sample concentration)

  • Endogenous peroxidase activity (add quenching step with 3% H₂O₂)

  • Protein aggregation (optimize sample preparation buffers)

• Technical factors:

  • Membrane contamination (handle membranes with clean forceps only)

  • Suboptimal blocking agent (try different blockers: milk, BSA, commercial blockers)

  • Detection system sensitivity (adjust exposure time or substrate concentration)

• Antibody-specific factors:

  • Too high antibody concentration (titrate to determine optimal dilution)

  • Antibody degradation (use fresh aliquots and avoid freeze-thaw cycles)

  • Polyclonal nature (consider affinity purification against the specific antigen)

Implementing a systematic approach to eliminate these issues one by one will help identify the specific cause of background in your experimental system .

How should I interpret contradictory results between ELISA and Western blot when using At3g07870 antibody?

When facing contradictory results between ELISA and Western blot using At3g07870 antibody, consider these analytical approaches:

• Analyze epitope accessibility differences:

  • ELISA detects native proteins while Western blot detects denatured proteins

  • Certain epitopes may be masked in native conformation but exposed after denaturation

  • Perform native PAGE Western blot as a comparative technique

• Evaluate technical parameters:

  • Sensitivity differences (ELISA typically more sensitive than Western blot)

  • Sample preparation variations (different buffers may affect protein conformation)

  • Antibody concentration optimization for each technique separately

• Consider protein post-translational modifications:

  • Phosphorylation or other modifications might affect antibody recognition

  • Run phosphatase-treated samples in parallel

  • Use modification-specific detection methods to identify potential PTMs

• Quantitative analysis approach:

  • Prepare standard curves using recombinant At3g07870 protein

  • Run spike-in recovery tests with known quantities of target protein

  • Implement statistical analysis to determine significance of differences

How can I differentiate between specific At3g07870 signal and cross-reactivity with similar plant proteins?

Differentiating specific At3g07870 signal from cross-reactivity requires:

• Comprehensive controls implementation:

  • Genetic knockout/knockdown controls lacking At3g07870

  • Heterologous expression systems with only At3g07870 present

  • Peptide competition assays using the immunizing peptide

• Advanced analytical techniques:

  • Immunoprecipitation followed by mass spectrometry identification

  • Two-dimensional electrophoresis to separate proteins by both pI and molecular weight

  • Super-resolution microscopy to confirm subcellular localization patterns

• Cross-species validation:

  • Test antibody against plant species with known sequence divergence in At3g07870

  • Create a gradient of relatedness to establish specificity boundaries

  • Express recombinant proteins with controlled sequence variations

• Bioinformatic analysis:

  • Conduct sequence alignment of At3g07870 with potential cross-reactive proteins

  • Predict epitopes using computational tools

  • Design custom peptide arrays covering potential cross-reactive epitopes

By systematically implementing these approaches, researchers can confidently distinguish between specific signals and cross-reactivity, especially in complex plant systems with highly conserved protein families .

How can I integrate At3g07870 antibody-based techniques with other omics approaches?

Integrating At3g07870 antibody techniques with omics approaches enables multi-dimensional insights:

• Proteomics integration:

  • Immunoprecipitate At3g07870 followed by mass spectrometry to identify interaction partners

  • Combine with BioID or APEX proximity labeling to map protein neighborhoods

  • Use antibody-based protein arrays to quantify across multiple conditions

• Transcriptomics correlation:

  • Correlate protein levels (detected by ELISA/Western blot) with mRNA expression data

  • Implement parallel RNAseq and protein quantification across development or stress conditions

  • Identify post-transcriptional regulation events by measuring protein:mRNA ratios

• Genomics applications:

  • Use ChIP-seq with At3g07870 antibody if the protein has DNA-binding properties

  • Correlate genetic variants with protein expression/modification patterns

  • Implement Mendelian randomization approaches to establish causality

• Metabolomics correlation:

  • Link At3g07870 protein levels with metabolite profiles

  • Establish potential enzymatic activities through metabolite changes in knockout vs. wild-type

  • Create multi-omics networks centered on At3g07870 function

This integrated approach provides a systems-level understanding of At3g07870's role in plant biology beyond what could be achieved with antibody-based techniques alone .

What are the best approaches for using At3g07870 antibody in plant stress response studies?

For studying plant stress responses using At3g07870 antibody:

• Experimental design considerations:

  • Include time-course sampling (0h, 1h, 3h, 6h, 12h, 24h, 48h)

  • Compare multiple stress types (drought, salinity, pathogen, temperature)

  • Use both whole-tissue and subcellular fractionation approaches

• Quantitative techniques:

  • Implement quantitative Western blotting with internal loading controls

  • Use ELISA for high-throughput screening across multiple conditions

  • Consider automated immunofluorescence image analysis for spatial information

• Stress-specific protocols:

  • For pathogen stress: Collect samples at specific infection stages

  • For abiotic stress: Control stress application precisely using controlled environments

  • For combined stresses: Design factorial experiments with appropriate controls

• Data analysis framework:

  • Normalize protein levels to unstressed controls

  • Apply statistical tests appropriate for time-series data

  • Create mathematical models correlating stress intensity with protein level changes

This comprehensive approach enables researchers to establish how At3g07870 protein levels, modifications, or localization may change during plant stress responses .

How can I apply biophysics-informed modeling to develop customized At3g07870 antibody variants with specific binding profiles?

Developing customized At3g07870 antibody variants with specific binding profiles requires:

• Structural characterization foundation:

  • Obtain structural data of antibody-antigen complex through X-ray crystallography or cryo-EM

  • Implement computational modeling if structural data is unavailable

  • Use molecular dynamics simulations to understand binding energetics

• Epitope mapping and engineering:

  • Identify key binding residues through alanine scanning mutagenesis

  • Design antibody variants with modifications at CDR3 regions

  • Use phage display technology to select for variants with desired specificity profiles

• High-throughput screening approach:

  • Create a library of antibody variants with systematic CDR modifications

  • Implement next-generation sequencing to analyze selection outcomes

  • Utilize machine learning to identify sequence-function relationships

• Validation framework:

  • Test predicted antibody variants using surface plasmon resonance

  • Verify cross-reactivity profiles against related antigens

  • Validate in relevant biological assays (Western blot, ELISA, etc.)

This biophysics-informed approach enables the development of tailored antibody variants that can distinguish between closely related epitopes, which is particularly valuable when studying protein families with high sequence homology in Arabidopsis research .

What are the detailed technical specifications for commercially available At3g07870 antibody?

The technical specifications for At3g07870 antibody include:

These detailed specifications help researchers plan experiments appropriately and ensure reproducibility across different research groups working with this antibody .

What protein-protein interaction networks have been established using At3g07870 antibody in Arabidopsis research?

Protein-protein interaction networks established using At3g07870 antibody reveal:

• Core interaction partners:

  • Several transcription factors have been identified as direct interactors

  • Components involved in cellular trafficking show consistent associations

  • Multiple proteins involved in developmental processes demonstrate interactions

• Network convergence patterns:

  • At3g07870 appears to connect with highly connected nodes in the Arabidopsis cellular network

  • Integration with interaction data from pathogen studies reveals potential roles in immunity

  • The protein shows association with both pathogen-specific and common host targets

• Technical approaches used:

  • Co-immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screens using At3g07870 as bait

  • Bimolecular fluorescence complementation to confirm interactions in planta

• Functional implications:

  • Network analysis suggests roles in stress response pathways

  • Integration with transcriptomic data indicates potential regulatory functions

  • Comparison with networks from other pathogens reveals potential converging virulence targets

Understanding these interaction networks provides crucial insights into At3g07870's biological function and its potential roles in plant immunity, development, and stress responses .