At2g17036 Antibody

What is At2g17036 and what role does it play in Arabidopsis?

At2g17036 is a gene locus in Arabidopsis thaliana that encodes a protein involved in auxin response pathways. The protein belongs to the Aux/IAA family, which are short-lived nuclear proteins that mediate auxin responses through protein-protein interactions. Research indicates these proteins may play significant roles in drought tolerance mechanisms through regulation of glucosinolate biosynthesis pathways . The antibody against this protein allows researchers to detect and quantify its expression, thereby facilitating studies of auxin signaling and stress responses in plants.

What applications is the At2g17036 antibody validated for?

The At2g17036 antibody has been validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications . These techniques allow researchers to:

• Quantify At2g17036 protein levels in plant tissue extracts (ELISA)

• Determine the molecular weight and confirm the identity of the protein (WB)

• Analyze expression patterns across different tissues or under varying experimental conditions

• Investigate post-translational modifications that may affect protein function

When designing experiments, researchers should follow established protocols for these techniques while incorporating appropriate positive and negative controls to ensure specificity.

What are the optimal storage conditions for the At2g17036 antibody?

For maximum stability and activity retention, the At2g17036 antibody should be stored at -20°C or -80°C immediately upon receipt . The antibody is provided in liquid form in a storage buffer containing 50% Glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . Repeated freeze-thaw cycles should be avoided as they can lead to antibody degradation and reduced performance. For short-term use (1-2 weeks), storage at 4°C is acceptable, but long-term storage requires freezing temperatures. Aliquoting the antibody upon receipt is recommended to minimize freeze-thaw cycles.

How specific is the At2g17036 antibody and what is its cross-reactivity profile?

The At2g17036 antibody demonstrates specific reactivity against Arabidopsis thaliana proteins . This polyclonal antibody was generated using a recombinant Arabidopsis thaliana At2g17036 protein as the immunogen . While primarily designed for Arabidopsis studies, researchers studying related plant species should perform validation tests to determine potential cross-reactivity with orthologous proteins. The antibody has undergone affinity purification to enhance specificity, though as with all polyclonal antibodies, some batch-to-batch variation may occur.

How should I design rigorous experiments using At2g17036 antibody?

A well-designed experiment using the At2g17036 antibody should incorporate principles of controlled experimental design. According to experimental research methodology best practices, your design should:

• Define clear research questions: Formulate specific hypotheses about At2g17036 function, expression, or regulation .

• Include proper controls:

  • Positive controls (samples known to express At2g17036)

  • Negative controls (samples lacking the target protein)

  • Loading controls (housekeeping proteins) for normalization

• Ensure adequate sample size: Calculate sample sizes based on expected effect sizes and desired statistical power .

• Minimize confounding variables: Control environmental factors and growth conditions that might affect At2g17036 expression.

• Randomize treatments: Assign experimental units to treatments randomly to avoid bias .

• Design appropriate treatment groups: Based on your research question, determine which variables to manipulate.

This approach aligns with sound experimental design principles, providing unbiased estimates of inputs and associated uncertainties while enabling detection of meaningful differences caused by independent variables .

What optimization steps are critical for Western blot analysis with At2g17036 antibody?

When optimizing Western blot protocols for At2g17036 detection, consider the following methodological steps:

• Sample preparation:

  • Extract proteins using buffers containing protease inhibitors

  • Determine optimal protein concentration (typically 20-50 μg per lane)

  • Include reducing agents if disulfide bonds may affect epitope accessibility

• Electrophoresis conditions:

  • Select appropriate gel percentage based on At2g17036's predicted molecular weight

  • Use fresh running buffer and maintain consistent voltage

• Transfer optimization:

  • Test different membrane types (PVDF vs. nitrocellulose)

  • Optimize transfer time and voltage for efficient protein transfer

• Blocking and antibody incubation:

  • Test multiple blocking agents (5% non-fat milk, BSA)

  • Determine optimal primary antibody dilution through titration experiments

  • Optimize incubation temperature and duration

• Detection method selection:

  • Choose between chemiluminescent, fluorescent, or colorimetric detection based on sensitivity requirements

  • Calibrate exposure times to avoid signal saturation

These methodological refinements can significantly improve detection sensitivity and specificity when working with the At2g17036 antibody.

How can I troubleshoot non-specific binding with the At2g17036 antibody?

Non-specific binding is a common challenge when working with antibodies. For the At2g17036 antibody, consider these methodological solutions:

• Increase blocking stringency:

  • Extend blocking time or increase blocking agent concentration

  • Add 0.1-0.3% Tween-20 to wash buffers to reduce hydrophobic interactions

• Optimize antibody concentration:

  • Perform titration experiments to find the minimum effective concentration

  • Dilute antibody in fresh blocking buffer containing 0.05% Tween-20

• Adjust incubation conditions:

  • Perform antibody incubations at 4°C overnight instead of room temperature

  • Increase washing duration and frequency between steps

• Pre-absorb the antibody:

  • Incubate diluted antibody with membrane containing non-target proteins

  • Remove antibodies that bind to common epitopes before using with your samples

• Validate with genetic controls:

  • If available, include samples from At2g17036 knockout plants as negative controls

  • Compare with overexpression lines as positive controls

These troubleshooting approaches can significantly improve signal-to-noise ratio and ensure reliable detection of the target protein.

What sample preparation techniques maximize At2g17036 detection in plant tissues?

Effective protein extraction from plant tissues requires specialized techniques to overcome challenges posed by cell walls, proteases, and interfering compounds:

• Tissue collection and processing:

  • Harvest tissues at consistent developmental stages

  • Flash-freeze samples in liquid nitrogen immediately after collection

  • Grind tissues to fine powder while maintaining frozen state

• Extraction buffer composition:

  • Include 1-2% PVPP to remove phenolic compounds

  • Add protease inhibitor cocktail to prevent degradation

  • Incorporate DTT or β-mercaptoethanol to maintain reducing conditions

  • Test different detergents (CHAPS, Triton X-100) for membrane protein solubilization

• Subcellular fractionation:

  • Consider nuclear extraction protocols if targeting nuclear pools of At2g17036

  • Use differential centrifugation to isolate relevant cellular compartments

• Protein concentration determination:

  • Use Bradford or BCA assays compatible with your extraction buffer

  • Ensure equal loading across samples for accurate comparisons

Proper sample preparation significantly impacts downstream detection and quantification of At2g17036 protein levels in experimental samples.

How can the At2g17036 antibody be used to study drought tolerance mechanisms?

Research indicates that Aux/IAA proteins like At2g17036 mediate drought tolerance in Arabidopsis . To investigate these mechanisms:

• Expression analysis under drought conditions:

  • Monitor At2g17036 protein levels via Western blot across a time course of water withholding

  • Compare expression between wild-type and drought-sensitive mutants

  • Correlate protein levels with physiological drought response parameters

• Protein-protein interaction studies:

  • Use co-immunoprecipitation with At2g17036 antibody to identify interaction partners during drought stress

  • Investigate associations with MYB28/29 transcription factors that regulate glucosinolate biosynthesis

  • Examine interactions with WRKY63, which has been implicated in drought response pathways

• Chromatin immunoprecipitation (ChIP) approaches:

  • Adapt ChIP-PCR protocols similar to those used for IAA19-YPet-His-FLAG

  • Investigate binding to auxin response elements in promoters of drought-responsive genes

  • Analyze how water stress affects DNA binding of complexes containing At2g17036

• Correlation with glucosinolate production:

  • Measure aliphatic glucosinolate levels in tissues with varying At2g17036 expression

  • Investigate relationships between At2g17036, MYB transcription factors, and glucosinolate biosynthesis genes

This multifaceted approach can reveal how At2g17036 contributes to plant drought tolerance through regulation of stomatal function and stress response pathways.

What approaches can elucidate At2g17036's role in auxin signaling pathways?

As a member of the Aux/IAA protein family, At2g17036 likely participates in auxin signaling through specific protein interactions. Advanced research approaches include:

• Quantitative co-localization studies:

  • Use the At2g17036 antibody alongside fluorescently-tagged ARF transcription factors

  • Analyze nuclear localization patterns under varying auxin concentrations

  • Quantify co-localization coefficients using confocal microscopy

• Protein degradation kinetics:

  • Measure At2g17036 protein stability following auxin treatment

  • Compare degradation rates between wild-type plants and auxin signaling mutants

  • Investigate post-translational modifications affecting protein stability

• Functional domain mapping:

  • Generate domain-specific antibodies or use epitope-tagged truncation constructs

  • Determine which protein regions mediate interactions with ARFs and TIR1/AFB auxin receptors

  • Correlate structural features with functional outcomes in auxin response assays

This comprehensive approach would provide mechanistic insights into how At2g17036 functions within the broader auxin signaling network to influence plant development and stress responses.

How can At2g17036 antibody be used to investigate crosstalk between hormone signaling pathways?

Plant hormone signaling pathways often exhibit complex crosstalk. The At2g17036 antibody can help investigate interactions between auxin and other hormones:

• Dual hormone treatment studies:

  • Analyze At2g17036 protein levels following combined treatments with auxin and ABA

  • Investigate changes in protein localization or modification status

  • Compare responses in wild-type vs. hormone signaling mutants

• Pathway interconnection analysis:

  • Examine At2g17036 expression in abi1 mutants deficient in ABA signaling

  • Investigate relationships between At2g17036, glucosinolate production, and stomatal regulation

  • Determine whether At2g17036 mediates auxin-ABA crosstalk during drought stress

• Transcription factor network mapping:

  • Use the antibody to identify associations with transcription factors from multiple hormone pathways

  • Investigate connections with WRKY63 and MYB28/29, which appear to function downstream of auxin signaling

  • Analyze how these interactions change under different stress conditions

This research would contribute to understanding how plants integrate multiple hormone signals to coordinate appropriate responses to environmental challenges.

What techniques can detect post-translational modifications of At2g17036?

Aux/IAA proteins are known to undergo various post-translational modifications that affect their function and stability. To investigate these modifications on At2g17036:

• Phosphorylation analysis:

  • Immunoprecipitate At2g17036 using the antibody followed by phospho-specific staining

  • Perform mass spectrometry to identify specific phosphorylated residues

  • Compare phosphorylation status under different treatment conditions

• Ubiquitination detection:

  • Use the antibody in combination with ubiquitin-specific antibodies in co-immunoprecipitation

  • Analyze ubiquitination patterns and kinetics after auxin treatment

  • Compare ubiquitination in TIR1/AFB mutant backgrounds

• SUMOylation studies:

  • Investigate potential SUMOylation of At2g17036 through immunoprecipitation and SUMO-specific detection

  • Determine how SUMOylation affects protein stability and interaction capabilities

  • Analyze changes in SUMOylation patterns under stress conditions

• PTM crosstalk analysis:

  • Develop a comprehensive map of multiple modifications on the same protein

  • Investigate whether certain modifications are prerequisites for others

  • Determine how modification patterns affect protein function

These approaches would provide critical insights into how post-translational modifications regulate At2g17036 activity and stability.

How should I quantify and normalize At2g17036 expression data?

Accurate quantification of protein expression requires careful normalization and statistical analysis:

• Densitometric analysis for Western blots:

  • Use software packages like ImageJ with gel analysis plugins

  • Ensure linear dynamic range by avoiding oversaturated signals

  • Normalize to appropriate loading controls (actin, tubulin, or total protein)

• ELISA data analysis:

  • Generate standard curves using purified recombinant At2g17036

  • Ensure samples fall within the linear range of detection

  • Calculate concentration based on 4 or 5-parameter logistic regression

• Biological replication and statistical approach:

  • Include at least 3-5 biological replicates per condition

  • Apply appropriate statistical tests (t-test, ANOVA)

  • Consider using mixed-effects models for complex experimental designs

This systematic approach ensures robust quantification of At2g17036 protein levels across experimental conditions.

How can I address data contradictions when studying At2g17036 expression?

Contradictory results can emerge when studying plant proteins like At2g17036 due to experimental variations. To resolve such contradictions:

• Systematic validation:

  • Verify antibody specificity using knockout mutants when available

  • Confirm results using complementary techniques (qRT-PCR, immunofluorescence)

  • Test whether contradictions arise from differences in growth conditions or developmental stages

• Experimental reconciliation strategy:

  • Identify key variables that differ between contradictory studies

  • Design experiments that systematically vary these parameters

  • Establish boundary conditions under which different outcomes occur

• Meta-analysis approach:

  • Aggregate data from multiple experiments or published studies

  • Identify patterns or dependencies that explain apparent contradictions

  • Develop integrative models that accommodate seemingly conflicting results

This methodical approach can transform apparent contradictions into deeper mechanistic insights about context-dependent protein functions.

What bioinformatic approaches can complement At2g17036 antibody studies?

Integrating experimental data with bioinformatic analyses provides comprehensive insights:

• Structural prediction and domain analysis:

  • Predict functional domains and regulatory motifs in At2g17036

  • Identify potential interaction interfaces and modification sites

  • Generate structural models to guide experimental design

• Network analysis:

  • Integrate protein interaction data with gene expression networks

  • Identify modules and pathways connected to At2g17036 function

  • Predict new functional relationships based on network positioning

• Phylogenetic analysis:

  • Compare At2g17036 sequences across plant species

  • Identify conserved regions that may indicate functional importance

  • Trace evolutionary relationships to inform functional studies

• Multi-omics data integration:

  • Correlate protein levels with transcriptomic and metabolomic data

  • Build predictive models incorporating multiple data types

  • Identify emergent properties not evident from single-omics approaches

These computational approaches enhance the interpretation of antibody-based studies and guide further experimental investigations.

How should I design experiments to study At2g17036 in relation to drought stress pathways?

To investigate At2g17036's role in drought responses, design experiments that systematically test its relationship with known drought response pathways:

• Genetic interaction studies:

  • Compare drought phenotypes between At2g17036 mutants and wild-type plants

  • Create double mutants with known drought response genes (e.g., WRKY63)

  • Analyze epistatic relationships to position At2g17036 within signaling cascades

• Pathway dissection approach:

  • Analyze protein expression in mutants with altered glucosinolate production (myb28 myb29)

  • Investigate connections with stomatal regulation pathways

  • Test interactions with both ABA-dependent and ABA-independent stress responses

• Systematic perturbation design:

  • Apply graduated levels of water stress to identify response thresholds

  • Combine drought with other stresses to test pathway specificity

  • Use chemical inhibitors to block specific signaling components

• Temporal dynamics analysis:

  • Design time-course experiments capturing both early and late responses

  • Monitor protein levels, modifications, and interactions at defined intervals

  • Correlate molecular changes with physiological drought response parameters

This experimental framework would provide comprehensive insights into At2g17036's functional role in drought stress responses.