At2g19060 Antibody

What is At2g19060 and why is an antibody against it useful in plant research?

At2g19060 refers to a gene locus in Arabidopsis thaliana with Uniprot number O64469. The antibody against this protein enables various molecular investigations including protein detection, localization studies, and protein-protein interaction analyses. This antibody is particularly valuable for researchers investigating molecular pathways in this important model plant species, potentially including studies related to iron metabolism based on contextual data .

For effective utilization, researchers should consider experimental design that incorporates appropriate controls, optimization of antibody concentration, and validation of specificity before conducting comprehensive studies of At2g19060 protein function and regulation.

What experimental techniques can be performed using the At2g19060 Antibody?

The At2g19060 Antibody can be employed in multiple research techniques:

• Western blotting/Immunoblot analysis: For detecting and quantifying At2g19060 protein in plant tissue extracts, allowing comparison of expression levels under different conditions .

• Immunoprecipitation (IP): To isolate At2g19060 protein complexes, enabling identification of interacting partners.

• Chromatin immunoprecipitation (ChIP): If At2g19060 functions as a DNA-binding protein or associates with chromatin, ChIP can identify its target DNA regions. The methodology described in search result outlines appropriate primer design and analysis approaches for ChIP-qPCR .

• Immunohistochemistry/Immunofluorescence: For visualizing protein localization at tissue, cellular, and subcellular levels.

• ELISA: For quantitative detection in plant samples, similar to the methodology described for antibody testing in search result .

Each application requires specific optimization, and researchers should conduct preliminary experiments to determine optimal antibody dilutions and experimental conditions.

What controls should be included when working with At2g19060 Antibody?

Proper experimental controls are essential for generating reliable data with At2g19060 Antibody:

• Positive controls:

  • Wild-type Arabidopsis samples expressing At2g19060

  • Recombinant At2g19060 protein (if available)

  • Overexpression lines of At2g19060

• Negative controls:

  • Knockout/knockdown lines of At2g19060

  • Non-specific IgG antibody controls for immunoprecipitation experiments

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

• Technical controls:

  • Loading controls (actin, tubulin) for western blots

  • Input samples for ChIP or IP experiments (typically 5-10% of starting material)

  • Concentration gradient tests to determine optimal antibody dilution

As noted in search result , immunoprecipitation experiments should include parallel samples with non-specific antibody (IgG) and the specific antibody to distinguish true signals from background .

How can At2g19060 Antibody be used to investigate protein-protein interactions in iron homeostasis pathways?

Based on the association between At2g19060 and research related to iron metabolism , the antibody can be employed to explore protein interaction networks through several methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use At2g19060 Antibody to pull down protein complexes from plant extracts

  • Analyze co-precipitated proteins via mass spectrometry

  • Validate interactions through reciprocal Co-IP experiments

  • Compare interaction profiles under iron-deficient versus iron-sufficient conditions

• Proximity-dependent labeling:

  • Fusion of At2g19060 with biotin ligase followed by streptavidin pulldown

  • Validation of interactions using At2g19060 Antibody

• Yeast two-hybrid confirmation:

  • Similar to the methodology described in search result showing "GRXS17 directly interacts with DRE2"

  • Use At2g19060 Antibody to confirm expression in mammalian systems

• Integration with transcriptome data:

  • Connect protein interaction data with transcriptional networks

  • Search result describes a "Coexpression network of up-regulated genes" approach that could be applied

Experimental design should include non-denaturing protein extraction conditions to preserve native interactions, and findings should be validated through multiple independent approaches.

What considerations are important for chromatin immunoprecipitation (ChIP) experiments using At2g19060 Antibody?

For researchers employing At2g19060 Antibody in ChIP studies:

• Chromatin preparation and crosslinking:

  • Optimize formaldehyde concentration (typically 1-3%) and crosslinking time

  • Verify chromatin fragmentation to achieve 200-500 bp fragments

  • Pre-clear chromatin with protein A/G beads to reduce background

• Immunoprecipitation parameters:

  • Determine optimal antibody concentration through titration experiments

  • Include appropriate controls as described in search result : "Each sample had two immunoprecipitations, one for the non-specific antibody (IgG) and the other for the anti HA-antibody (specific)"

  • Reserve 5-10% of starting chromatin as input control

• PCR primer design:

  • Design primers for potential binding regions following guidelines in "Primer design for ChIP"

  • Include negative control regions (typically intergenic regions)

  • Verify primer efficiency (90-110%) and specificity

• Data analysis:

  • Employ percent input method or fold enrichment over IgG for quantification

  • Analyze results according to methodologies outlined in "Analysis of ChIP-qPCR"

  • Apply appropriate statistical tests to determine significance

If investigating relationships with iron metabolism, consider examining binding near genes involved in iron homeostasis pathways and validating findings through multiple biological replicates.

How can researchers correlate At2g19060 protein levels with iron homeostasis phenotypes?

To establish functional relationships between At2g19060 and iron homeostasis:

• Experimental design:

  • Grow Arabidopsis under controlled iron-sufficient and iron-deficient conditions

  • Include time course sampling to capture dynamic responses

  • Compare wild-type plants with relevant mutants (similar to grxs17 mutants described in search result )

• Protein and transcript analysis:

  • Quantify At2g19060 protein using the antibody in western blots

  • Measure transcript levels via RT-qPCR following protocols described in search result

  • Correlate protein expression with transcript abundance to identify post-transcriptional regulation

• Iron status verification:

  • Employ "Iron reductase assay" methodology as described in search result

  • Use "Pearl stain for iron visualization" to assess iron distribution

  • These complementary approaches confirm iron status and localization

• Phenotypic characterization:

  • Document morphological responses to iron availability

  • Measure physiological parameters like chlorophyll content or photosynthetic efficiency

  • Assess molecular responses using marker genes for iron deficiency

• Data integration:

  • Correlate At2g19060 protein levels with phenotypic severity

  • Compare with published datasets on iron deficiency responses

  • Develop models of At2g19060 function in iron homeostasis networks

This comprehensive approach allows positioning At2g19060 within the broader context of iron regulatory networks in Arabidopsis.

What is the optimal protocol for western blot analysis using At2g19060 Antibody?

For reliable western blot detection of At2g19060:

• Sample preparation:

  • Harvest and flash-freeze plant tissue in liquid nitrogen

  • Grind to fine powder and extract proteins in buffer containing:

    • 50 mM Tris-HCl pH 7.5

    • 150 mM NaCl

    • 1% Triton X-100

    • 1 mM EDTA

    • Protease inhibitor cocktail

  • Centrifuge at 14,000 × g, 15 min, 4°C and quantify protein concentration

• SDS-PAGE and transfer:

  • Separate 20-30 μg protein on 10-12% SDS-PAGE gel

  • Transfer to PVDF or nitrocellulose membrane

  • Verify transfer with Ponceau S staining

• Immunodetection:

  • Block with 5% non-fat dry milk in TBST for 1 hour

  • Incubate with At2g19060 Antibody at 1:1000 to 1:5000 dilution (based on similar dilution ranges mentioned in search result )

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3 × 10 min with TBST

  • Incubate with HRP-conjugated secondary antibody (1:10,000) for 1 hour

  • Wash 3 × 10 min with TBST and develop using ECL substrate

• Controls and quantification:

  • Include molecular weight marker and appropriate controls

  • Reprobe with antibody against housekeeping protein for normalization

  • Perform densitometry analysis on biological replicates (n≥3)

This protocol provides a starting point that should be optimized for specific experimental conditions and antibody lot characteristics.

How should researchers design experiments to investigate At2g19060 protein localization?

To determine At2g19060 subcellular localization:

• Immunofluorescence approach:

  • Fix Arabidopsis tissues with 4% paraformaldehyde

  • Permeabilize with 0.1-0.5% Triton X-100

  • Block with 3% BSA and incubate with At2g19060 Antibody (1:100-1:500)

  • Visualize using fluorophore-conjugated secondary antibody

  • Include DAPI nuclear stain and organelle-specific markers

  • Image using confocal microscopy

• Subcellular fractionation:

  • Isolate subcellular compartments (cytosolic, nuclear, chloroplastic, mitochondrial)

  • Verify fraction purity using established marker proteins

  • Perform western blotting with At2g19060 Antibody on each fraction

  • Quantify distribution across compartments

• Validation approaches:

  • Compare with GFP-tagged At2g19060 localization patterns

  • Examine co-localization with known iron homeostasis proteins

  • Assess potential relocalization under stress conditions

Since search result indicates potential relationships with cytosolic iron metabolism through GRXS17 associations , researchers should examine whether At2g19060 exhibits cytosolic localization or associates with specific organelles involved in iron homeostasis.

What methods should be employed to validate At2g19060 Antibody specificity?

Rigorous antibody validation is essential for reliable research outcomes:

• Western blot validation:

  • Compare signal between wild-type and knockout/knockdown lines

  • Verify detection of a single band at the expected molecular weight

  • Conduct peptide competition assay by pre-incubating antibody with immunizing peptide

• Immunoprecipitation-Mass Spectrometry:

  • Perform IP with At2g19060 Antibody and analyze by MS

  • Compare with IgG control immunoprecipitation

  • Confirm pulldown of correct target protein

• Heterologous expression:

  • Express recombinant At2g19060 in bacterial or yeast systems

  • Test antibody detection in expression system versus controls

  • Assess cross-reactivity with related proteins

• Epitope mapping:

  • Determine which region of At2g19060 the antibody recognizes

  • Assess potential cross-reactivity based on sequence homology

  • Express protein fragments to confirm epitope specificity

• Cross-validation:

  • Compare protein detection with transcript expression patterns

  • Validate localization using complementary approaches

  • Test multiple antibody lots if available

The inclusion of proper controls, as emphasized in search result regarding non-specific antibody (IgG) controls , is essential for distinguishing specific from non-specific signals in all validation experiments.

What are common challenges when using At2g19060 Antibody and how can they be addressed?

Researchers may encounter several issues when working with At2g19060 Antibody:

• No signal detected:

  • Increase antibody concentration (try 1:500 dilution)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Increase protein loading (up to 50 μg)

  • Use more sensitive detection methods

  • Verify target protein expression in samples

• Multiple bands or high background:

  • Optimize blocking conditions (increase to 5% BSA)

  • Increase antibody dilution (1:5000 or higher)

  • Extend washing steps (5 × 10 min TBST washes)

  • Use freshly prepared samples to avoid degradation products

  • Consider adding phosphatase inhibitors if phosphorylation affects recognition

• Inconsistent results:

  • Standardize protein extraction methodology

  • Process samples in parallel under identical conditions

  • Verify equal loading with Ponceau S and housekeeping controls

  • Prepare working solutions in larger batches

  • Control incubation times and temperatures precisely

• Detection in complex samples:

  • Consider enrichment by immunoprecipitation before detection

  • Use more sensitive ECL substrates

  • Optimize protein extraction buffer components

  • Test alternative membrane types (PVDF vs. nitrocellulose)

When optimizing antibody concentration, search result notes that "3 μl antibody (~4 μg) antibody (IgG or anti HA)" was used in their immunoprecipitation experiments, which may provide a reference point for initial testing .

How can researchers integrate At2g19060 protein data with transcriptome analysis?

For comprehensive understanding of At2g19060 function:

• Multi-omics experimental design:

  • Collect parallel samples for protein analysis and RNA extraction

  • Include multiple timepoints and treatment conditions

  • Ensure adequate biological replication for statistical validity

• Transcriptome analysis:

  • Perform RNA-seq or microarray analysis following methodologies in "Transcriptome analysis" from search result

  • Identify genes differentially expressed in response to At2g19060 manipulation

  • Construct co-expression networks similar to the approach described in search result

• Protein-transcript correlation:

  • Quantify At2g19060 protein levels via western blot using the antibody

  • Compare with corresponding transcript levels via qRT-PCR

  • Identify conditions where protein and transcript levels diverge (suggesting post-transcriptional regulation)

• ChIP integration (if applicable):

  • If At2g19060 functions in transcriptional regulation, perform ChIP using methodologies described in search result

  • Correlate binding sites with expression changes

  • Identify potential regulatory targets

• Network analysis:

  • Construct integrated networks incorporating protein-level and transcript-level data

  • Identify modules and pathways affected by At2g19060

  • Generate testable hypotheses about At2g19060 function

This integrative approach positions At2g19060 within its broader biological context and reveals potential regulatory roles.

What statistical approaches should be used for analyzing ChIP-qPCR data with At2g19060 Antibody?

For robust analysis of ChIP-qPCR experiments:

• Data normalization methods:

  • Percent input method: Calculate enrichment as percentage of input chromatin

  • Fold enrichment over IgG: Compare specific antibody pulldown to non-specific IgG control

  • Reference gene normalization: Normalize to genes known not to be targets

  • Follow specific recommendations in "Analysis of ChIP-qPCR" from search result

• Technical considerations:

  • Run technical triplicates for each qPCR reaction

  • Ensure primer efficiency between 90-110%

  • Include no-template controls and melting curve analysis

  • Calibrate with standard curves for absolute quantification

• Statistical testing:

  • Apply Student's t-test for comparing two conditions

  • Use ANOVA for multiple condition comparisons

  • Implement multiple testing correction (Bonferroni or FDR)

  • Consider non-parametric tests if data distribution is non-normal

• Advanced analysis:

  • Correlation analysis between binding and gene expression

  • Motif discovery in enriched regions

  • Comparative analysis across experimental conditions

  • Integration with published ChIP datasets

• Visualization approaches:

  • Bar graphs showing fold enrichment with error bars

  • Genome browser tracks for contextualizing binding sites

  • Heatmaps for comparing binding patterns across multiple regions

Proper experimental design should include a minimum of three biological replicates and follow the methodological guidelines outlined in search result .

How should researchers interpret contradictory results between At2g19060 protein levels and phenotypic observations?

When protein data and phenotypic results don't align as expected:

• Systematic troubleshooting:

  • Verify antibody specificity using methods described in Question 3.3

  • Investigate post-translational modifications that may affect protein function without changing levels

  • Consider temporal dynamics: protein levels may change rapidly while phenotypes develop gradually

  • Examine spatial patterns: tissue-specific effects may be masked in whole-plant analyses

• Functional redundancy assessment:

  • Identify homologs or proteins with overlapping functions

  • Create multiple mutants to overcome redundancy

  • Test overexpression to determine if higher protein levels enhance phenotypes

• Pathway analysis:

  • Consider At2g19060's position in regulatory networks

  • Examine upstream regulators and downstream effectors

  • Based on context in search result , if related to iron metabolism, measure iron content and iron-dependent processes

• Environmental factors:

  • Test multiple growth conditions

  • Control for stress factors that might influence results

  • Consider developmental timing and circadian regulation

• Dosage effects:

  • Determine if there's a threshold effect for phenotype manifestation

  • Use quantitative approaches to correlate protein levels with phenotypic severity

  • Consider using more sensitive phenotyping methods

This systematic approach helps resolve apparent contradictions between molecular and phenotypic data.

What are the best practices for using At2g19060 Antibody in comparative studies across different Arabidopsis ecotypes?

When extending research to multiple Arabidopsis ecotypes:

• Sequence variation analysis:

  • Check sequence conservation of At2g19060 across ecotypes

  • Identify amino acid variations that might affect antibody recognition

  • Consider designing ecotype-specific antibodies if significant variation exists

• Experimental design:

  • Include standard ecotypes (Col-0, Ler, Ws) alongside specialized ecotypes

  • Standardize growth conditions precisely

  • Process all samples in parallel to minimize technical variation

  • Implement blocking experimental design to control for batch effects

• Validation requirements:

  • Test antibody specificity separately for each ecotype

  • Use recombinant proteins from different ecotypes to calibrate detection sensitivity

  • Include appropriate controls for each ecotype

• Data normalization strategies:

  • Use highly conserved housekeeping proteins as loading controls

  • Consider relative quantification rather than absolute comparisons

  • Validate protein detection with transcript analysis

• Interpretation framework:

  • Associate protein level differences with known phenotypic variations

  • Consider natural variation in iron homeostasis traits

  • Investigate correlations between protein levels and ecotype-specific adaptations

This approach ensures that comparative studies produce biologically meaningful insights rather than technical artifacts.

How can At2g19060 Antibody be incorporated into studies of plant responses to environmental stresses?

For investigating At2g19060's role in stress responses:

• Stress treatment experimental design:

  • Apply relevant stresses: iron deficiency/excess, oxidative stress, drought, heat

  • Include time course sampling to capture dynamic responses

  • Consider combination stresses to mimic natural conditions

  • Based on search result , iron-related stresses may be particularly relevant

• Multi-level analysis:

  • Monitor At2g19060 protein levels using the antibody during stress progression

  • Track potential subcellular relocalization using immunofluorescence

  • Identify stress-dependent protein interactions using co-immunoprecipitation

  • Measure downstream physiological parameters

• Complementary assays:

  • "Iron reductase assay" to measure iron acquisition capacity

  • "Pearl stain for iron visualization" to assess iron distribution

  • "Hydrogen peroxide Assay" to quantify oxidative stress

  • "UV detection of phenolic compounds" to evaluate secondary metabolism responses

• Genetic approaches:

  • Compare wild-type, knockout, and overexpression lines under stress conditions

  • Use inducible expression systems for temporal control

  • Create reporter lines to track both protein expression and activity

• Network integration:

  • Connect At2g19060 to known stress response pathways

  • Search result suggests potential connections to glutaredoxin GRXS17 and iron homeostasis

  • Consider relationships with iron-sulfur cluster assembly, which is often affected by oxidative stress

This integrated approach positions At2g19060 within plant stress response networks and reveals its specific contributions to stress adaptation mechanisms.