At5g66910 Antibody

What is AT5G66910 and why are antibodies against it important for plant research?

AT5G66910 is a gene locus in Arabidopsis thaliana, a model organism widely used in plant molecular biology research. Antibodies targeting the protein encoded by this gene are valuable tools for investigating protein localization, expression levels, and functional relationships in plant development studies. These antibodies enable researchers to track the presence and behavior of the protein in different tissues, under various conditions, and in different genetic backgrounds. In the post-genomic era, protein localization studies at subcellular, cellular, and tissue levels contribute significantly to our understanding of protein function, cell dynamics, protein-protein interactions, and regulatory networks . Antibodies against AT5G66910, like other protein-specific antibodies, serve as critical tools for advancing our understanding of plant biology through techniques such as western blot detection, immunolocalization, affinity purification, and chromatin immunoprecipitation .

What approaches are used to generate antibodies against Arabidopsis proteins like AT5G66910?

Two primary approaches are employed to generate antibodies against Arabidopsis proteins including AT5G66910:

How can researchers access antibodies for AT5G66910 and other Arabidopsis proteins?

Researchers interested in obtaining antibodies against AT5G66910 and other Arabidopsis proteins have several options:

• Use established community resources: The Centre for Plant Integrative Biology (CPIB) has developed antibodies against key Arabidopsis root proteins that are available through the Nottingham Arabidopsis Stock Centre . This collection includes antibodies against proteins involved in hormone synthesis, transport and perception, membrane trafficking, and subcellular markers .

• Commercial sources: Various commercial suppliers produce antibodies against plant proteins, though availability for specific targets like AT5G66910 may vary.

• Custom antibody production: Researchers can commission custom antibody production through commercial services or institutional facilities, following bioinformatic analysis to identify potential antigenic regions with minimal cross-reactivity .
  For all options, researchers should verify antibody specificity before use in critical experiments, ideally by testing in corresponding mutant backgrounds .

What bioinformatic approaches should be used to design effective antibodies against AT5G66910?

Designing effective antibodies against AT5G66910 requires a systematic bioinformatic approach:

• Identify potential antigenic regions: Use epitope prediction software to identify sections of the protein sequence with high antigenicity scores. Focus on regions with high surface probability, hydrophilicity, and flexibility .

• Assess cross-reactivity potential: For each candidate antigenic region, perform database searches using BlastX to check for sequence similarity with other proteins. The CPIB project used a threshold of less than 40% amino acid similarity to minimize cross-reactivity .

• Select optimal region size: For recombinant protein antigens, aim for approximately 100 amino acids. If high similarity regions are identified, use a sliding window approach to find a smaller subsequence with reduced similarity .

• Consider protein structure: Where possible, avoid transmembrane domains and regions critical for protein folding.

• Multiple sequence alignment: If targeting a specific family member (like AT5G66910), align with related proteins to identify unique regions.
  This systematic bioinformatic pipeline has proven robust for antibody development against Arabidopsis proteins, as demonstrated by the CPIB antibody project's 55% success rate with recombinant protein antibodies .

What are the best validation methods to confirm specificity of AT5G66910 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For AT5G66910 antibodies, several complementary approaches are recommended:

• Testing in null mutant backgrounds: The most rigorous validation method is comparing antibody signal between wild-type plants and null mutants of AT5G66910. Absence of signal in the mutant strongly confirms specificity, as demonstrated with multiple Arabidopsis antibodies in the CPIB project .

• Western blot analysis: Verify that the antibody detects a single band of the expected molecular weight. The calculated molecular weight of the protein can be compared with observed band size, though post-translational modifications and protein hydrophobicity can affect migration patterns .

• Dot blot titration: Assess antibody sensitivity by testing serial dilutions of the recombinant protein. High-quality antibodies typically detect target proteins in the picogram range .

• Immunolocalization patterns: Compare observed localization patterns with known or predicted subcellular localization data. Consistency with published localization data (if available) or predictions supports antibody validity .

• Pre-adsorption controls: Incubate antibodies with excess antigen before immunostaining to confirm signal specificity.
  The CPIB project found that multiple antibodies against Arabidopsis proteins showed no detectable signal in respective mutant backgrounds by immunolocalization, confirming their specificity .

What purification methods improve the performance of antibodies for AT5G66910 detection?

Antibody purification significantly impacts detection quality. For AT5G66910 antibodies, the following purification methods should be considered:

• Affinity purification: The most effective method is affinity purification against the purified recombinant protein. The CPIB project demonstrated that this approach dramatically improved detection rates from near zero to 55% of antibodies showing high-confidence signals .

• Protein A/G purification: Standard purification methods using Protein A or Protein G can help remove serum proteins but are less effective than antigen-specific affinity purification .

• Caprylic acid precipitation: This method can help purify IgG from serum but showed limited improvement in detection rates compared to affinity purification in the CPIB project .

• Signal amplification methods: Though not strictly purification techniques, signal amplification protocols may enhance detection for low-abundance proteins, but they did not significantly improve detection rates in the CPIB study compared to affinity purification .
  The data from the CPIB antibody project clearly demonstrates that affinity purification against the target protein is the most effective approach for enhancing antibody performance in both immunolocalization and western blot applications .

Why might my AT5G66910 antibody fail to detect signal in immunolocalization experiments?

Several factors could explain why an AT5G66910 antibody might fail to detect signal in immunolocalization experiments:

• Low protein abundance: The AT5G66910 protein may be expressed at levels below detection limits. Even with high-quality affinity-purified antibodies, the CPIB project found that some antibodies failed to produce signals despite good purification quality .

• Insufficient antibody purification: Crude antisera often fail to produce detectable signals in immunolocalization. The CPIB project found that most crude antibodies showed no signal when tested by in situ immunolocalization, with only a few exceptions (PIN1, PIN2, PIN3, PIN4, PIN7 and PM-ATPase) .

• Epitope masking: Protein interactions or conformational changes may conceal the epitope in the cellular context.

• Fixation-induced epitope destruction: Some fixation protocols may alter protein structure and destroy antibody recognition sites.

• Inappropriate tissue preparation: Poor tissue penetration or excessive background can mask specific signals.

• Low antibody specificity or titer: Despite good affinity purification, some animals produce poor immune responses resulting in low levels of specific IgG .
  The CPIB project found that affinity purification was crucial for improving detection rates, but even after this treatment, some antibodies still failed to detect signals in immunolocalization experiments .

How can I resolve western blot issues when working with AT5G66910 antibodies?

When facing challenges with western blot detection of AT5G66910, consider these troubleshooting approaches:

• Unexpected band sizes: For membrane proteins like those involved in signaling pathways, hydrophobic nature may cause abnormal migration on gels. The CPIB project noted that for some antibodies, detected band sizes differed from predicted molecular weights .

• Multiple bands: These could indicate degradation products, post-translational modifications, or cross-reactivity. Verify specificity by testing in the corresponding mutant background .

• No signal detection: Consider:

  • Increasing protein loading amount

  • Reducing the antibody dilution

  • Extending incubation times

  • Using more sensitive detection systems

  • Optimizing transfer conditions for membrane proteins

  • Trying different protein extraction methods to improve solubilization

• High background: Improve blocking conditions, increase washing steps, and further purify antibodies. The CPIB study showed that affinity purification significantly improved signal-to-noise ratio compared to crude antisera .

• Protein extraction optimization: For membrane or low-abundance proteins, specialized extraction buffers may be required to maintain protein integrity and improve detection.
  The CPIB project validated several antibodies by western blot against their respective mutant backgrounds, confirming specificity through the absence of bands in mutant samples .

What controls should be included when using AT5G66910 antibodies for experimental validation?

Rigorous controls are essential for reliable antibody-based experiments:

• Genetic controls:

  • Wild-type tissue as positive control

  • Null mutant of AT5G66910 as negative control

  • Overexpression lines as positive controls with enhanced signal

  • The CPIB project demonstrated the value of mutant controls, showing complete absence of signal in mutants for most tested antibodies

• Technical controls:

  • Pre-immune serum control to evaluate background

  • Secondary antibody-only control to assess non-specific binding

  • Dot blot titration to determine detection sensitivity

  • Peptide competition assay where excess antigen blocks specific binding

• Validation across methods:

  • Compare results between immunolocalization and western blot

  • Confirm localization with fluorescent protein fusions

  • Correlate protein detection with transcript levels from RT-PCR

• Cross-validation with other antibodies:

  • If available, compare results with commercial antibodies

  • Use subcellular marker antibodies for co-localization studies
    The CPIB antibody resource validated antibodies against subcellular markers (BIP, γ-cop, PM-ATPase, and MDH) that can serve as valuable controls for co-localization experiments with AT5G66910 antibodies .

How can AT5G66910 antibodies be used to study protein-protein interactions and regulatory networks?

AT5G66910 antibodies can be leveraged for several advanced applications to study protein interactions and regulatory networks:

• Co-immunoprecipitation (Co-IP):

  • Use affinity-purified AT5G66910 antibodies to pull down the target protein along with its interacting partners

  • Identify novel interaction partners through mass spectrometry analysis of precipitated complexes

  • Confirm specific interactions by western blot analysis of precipitated material

• Chromatin Immunoprecipitation (ChIP):

  • If AT5G66910 encodes a DNA-binding protein, ChIP can identify genomic binding sites

  • ChIP-seq combines this approach with next-generation sequencing to map genome-wide binding sites

  • The CPIB antibody collection specifically mentions ChIP as one of the applications for their antibodies

• Proximity-dependent labeling:

  • Combine antibody-based detection with BioID or APEX2 proximity labeling to identify proteins in the same subcellular neighborhood

• Protein complex analysis:

  • Use antibodies to track AT5G66910 through size-exclusion chromatography to identify complex formation

  • Blue native PAGE followed by western blotting can preserve and detect native protein complexes

• Dynamic protein studies:

  • Combine with cellular fractionation to track protein movement between compartments

  • Use in pulse-chase experiments to study protein turnover rates
    The CPIB study emphasized that better understanding of protein localization contributes to understanding protein-protein interactions and regulatory networks, highlighting the value of high-quality antibodies for these applications .

What approaches can be used to study AT5G66910 protein localization at subcellular, cellular, and tissue levels?

Understanding AT5G66910 protein localization across different biological scales requires multi-faceted approaches:

• Subcellular localization:

  • Immunogold electron microscopy for high-resolution localization

  • Co-localization with established subcellular markers (e.g., BIP for ER, γ-cop for Golgi, PM-ATPase for plasma membrane, and MDH for plastids)

  • Super-resolution microscopy techniques like STORM or PALM for detailed subcellular distribution

  • Subcellular fractionation followed by western blotting

• Cellular-level analysis:

  • Immunohistochemistry on tissue sections to determine cell-type specific expression

  • Whole-mount immunofluorescence for intact tissue analysis

  • The CPIB project demonstrated successful immunolocalization for 22 out of 38 antibodies

• Tissue-level distribution:

  • Tissue-specific western blot analysis to compare expression levels

  • Immunohistochemistry across developmental stages

  • Comparison of expression between different organs

• Dynamic localization studies:

  • Track protein relocation in response to stimuli or stresses

  • Study developmental changes in protein distribution

  • Compare localization in different mutant backgrounds to understand regulatory relationships
    The CPIB antibody project specifically noted that better understanding of protein localization at subcellular, cellular, and tissue levels contributes to understanding protein function and role in cell dynamics , making these approaches particularly valuable for AT5G66910 research.

How can AT5G66910 antibodies be used to study protein modifications and processing?

AT5G66910 antibodies can be powerful tools for investigating post-translational modifications and protein processing:

• Detecting protein modifications:

  • Western blot analysis to identify mobility shifts indicating modifications

  • Combined use of AT5G66910 antibodies with modification-specific antibodies (phospho-specific, ubiquitin, SUMO, etc.)

  • Immunoprecipitation followed by mass spectrometry to identify specific modifications

• Monitoring protein processing:

  • Use antibodies targeting different domains to track proteolytic processing

  • Compare band patterns between tissues or conditions to identify differential processing

  • Pulse-chase experiments with immunoprecipitation to track protein maturation

• Studying modification dynamics:

  • Compare modification status under different conditions or treatments

  • Track changes in modification patterns during development

  • Analyze modification differences between wild-type and mutant plants

• Modification site mapping:

  • Immunoprecipitate AT5G66910 protein and analyze by mass spectrometry

  • Compare modification patterns with predicted sites from bioinformatic analysis

  • Use site-directed mutagenesis to confirm functional significance of modifications

• Conformational analysis:

  • Employ conformation-specific antibodies to detect structural changes

  • Study effects of modifications on protein conformation through differential antibody recognition
    The CPIB study noted that some antibodies detected bands different from expected sizes in western blots, which could be attributed to post-translational modifications , highlighting the utility of antibodies in studying these processes.

How can I integrate AT5G66910 antibody data with other systems biology approaches?

Integrating antibody-derived data with other systems biology approaches creates a more comprehensive understanding of AT5G66910 function:

• Transcriptomics integration:

  • Compare protein levels (western blot) with transcript levels (RNA-seq/qPCR)

  • Identify discrepancies suggesting post-transcriptional regulation

  • The CPIB emphasizes integrative systems biology approaches for understanding protein function

• Proteomics correlation:

  • Compare antibody-detected localization with proteomics-derived subcellular fractionation data

  • Correlate protein abundance changes with global proteomics datasets

  • Validate mass spectrometry-identified interactions with co-immunoprecipitation

• Metabolomics connections:

  • Correlate protein levels or modifications with metabolite changes

  • Link protein localization changes to metabolic pathway alterations

• Phenomics relationships:

  • Correlate protein expression patterns with phenotypic data from AT5G66910 mutants

  • Map protein localization changes to developmental phenotypes

• Computational modeling:

  • Incorporate protein localization and interaction data into cellular models

  • Use protein dynamics data to inform systems-level simulations

  • The CPIB specifically mentions the value of protein localization data for modeling multi-cellular systems using integrative systems biology approaches

• Data visualization and integration tools:

  • Use pathway mapping tools to place AT5G66910 in biological context

  • Apply network analysis to position the protein within interaction networks

  • Create multi-layered visualizations incorporating transcriptomic, proteomic, and localization data
    The CPIB antibody project was specifically designed to support integrative systems biology approaches to understand root development, demonstrating the value of antibody-derived data in comprehensive biological analysis .

What are the best practices for quantifying AT5G66910 protein levels across different experimental conditions?

Accurate quantification of AT5G66910 protein levels requires rigorous methodological approaches:

• Normalization approaches:

  • Use multiple housekeeping proteins as loading controls

  • Consider tissue-specific reference proteins

  • Normalize to total protein methods (e.g., Ponceau staining)

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Use sufficient biological and technical replicates (minimum n=3)

  • Consider power analysis to determine sample size

• Validation strategies:

  • Confirm findings with orthogonal methods

  • Verify with genetic controls (overexpression, knockdown)

  • The CPIB project validated antibodies against multiple genetic backgrounds

• Standardization protocols:

  • Develop standard operating procedures for sample preparation

  • Use consistent extraction buffers optimized for the protein

  • Implement quality control metrics for each experiment
    The CPIB antibody project noted that affinity purification significantly improved detection quality, suggesting this as an essential step for quantitative applications .

How can I address contradictory results between antibody-based detection and other experimental approaches when studying AT5G66910?

Resolving contradictions between antibody-based results and other experimental approaches requires systematic troubleshooting:

• Identify the nature of the contradiction:

  • Localization discrepancies (e.g., antibody vs. fluorescent fusion protein)

  • Expression level inconsistencies (e.g., western blot vs. transcript levels)

  • Functional outcomes (e.g., antibody blocking vs. genetic knockout)

• Evaluate antibody reliability:

  • Reconfirm antibody specificity in appropriate controls

  • Test in null mutant backgrounds to verify absence of signal

  • Validate with additional antibodies targeting different epitopes

  • The CPIB study emphasized the importance of validating antibodies against mutant backgrounds

• Assess technical limitations:

  • Consider detection limits of each method

  • Evaluate tissue preparation effects on epitope accessibility

  • Examine temporal aspects (protein vs. mRNA stability differences)

  • Consider post-translational modifications affecting antibody recognition

• Reconciliation approaches:

  • Design experiments to directly compare methods

  • Use complementary approaches to address limitations of each method

  • Consider biological reasons for discrepancies (e.g., post-transcriptional regulation)

• Systematic documentation:

  • Document all experimental conditions thoroughly

  • Report contradictory results transparently in publications

  • Discuss limitations of each approach
    The CPIB antibody project noted that despite good quality affinity purification, some antibodies failed to detect signals, suggesting that contradictory results might stem from technical limitations rather than antibody quality issues .

What are the best approaches for using AT5G66910 antibodies in cross-species studies?

Using AT5G66910 antibodies across different plant species requires careful consideration:

What are the most advanced applications of AT5G66910 antibodies in plant developmental biology research?

AT5G66910 antibodies enable several cutting-edge applications in plant developmental biology:

• Live tissue dynamics:

  • Use cell-permeable fluorescent-labeled antibody fragments to track protein dynamics in living tissues

  • Combine with light-sheet microscopy for 4D protein tracking during development

• Single-cell protein analysis:

  • Apply antibodies in single-cell proteomics workflows

  • Combine with laser capture microdissection for cell-type-specific protein profiles

  • The CPIB project emphasized the importance of understanding protein localization at cellular and tissue levels

• Developmental proteomics:

  • Track protein modifications across developmental stages

  • Map protein interaction networks during tissue differentiation

  • Correlate with developmental transcriptomics data

• Hormone response studies:

  • Investigate protein relocalization in response to hormone treatments

  • Study post-translational modifications induced by hormonal signaling

  • The CPIB antibody collection includes antibodies against key proteins involved in hormone synthesis, transport, and perception

• Environmental response analysis:

  • Monitor protein behavior under various stresses

  • Track changes in protein-protein interactions during adaptation responses

  • Map protein modifications in response to environmental signals

• Tissue-specific interactome mapping:

  • Use antibodies for tissue-specific protein complex isolation

  • Compare interaction partners across developmental stages

  • Identify tissue-specific regulatory mechanisms
    The CPIB antibody project was specifically developed to support research on root development in Arabidopsis, highlighting the value of antibodies in developmental biology research .

How can computational approaches enhance the utility of AT5G66910 antibody data in research?

Computational approaches significantly enhance the value of antibody-derived data:

• Epitope prediction and antibody design:

  • Machine learning algorithms improve epitope prediction accuracy

  • Structure-based computational approaches for optimal antibody design

  • The CPIB project used bioinformatic analysis to identify antigenic regions and minimize cross-reactivity

• Image analysis automation:

  • Develop deep learning models for automated quantification of immunolocalization

  • Apply computer vision techniques for high-throughput analysis

  • Create 3D reconstruction algorithms for spatial protein distribution

• Network analysis and visualization:

  • Integrate antibody-derived interaction data into protein-protein interaction networks

  • Apply graph theory to identify key nodes and regulatory hubs

  • Develop multi-layer network visualizations incorporating localization data

• Predictive modeling:

  • Use protein localization data to inform cellular computational models

  • Develop predictive algorithms for protein behavior under various conditions

  • The CPIB emphasized the value of protein localization data for modeling multi-cellular systems

• Multi-omics data integration:

  • Develop computational frameworks to integrate antibody-derived protein data with transcriptomics, metabolomics, and phenomics

  • Apply machine learning for pattern recognition across multi-modal datasets

  • Create predictive models incorporating protein dynamics

• Database development and data mining:

  • Create searchable repositories of antibody-derived localization data

  • Develop computational tools to mine existing datasets for new insights

  • Implement standardized metadata for improved cross-study comparisons
    The CPIB project noted that their antibody resource was developed to support integrative systems biology approaches, highlighting the importance of computational methods in maximizing the value of antibody data .