ATJ2 Antibody

What is ATJ2 Antibody and what does it target in Arabidopsis?

ATJ2 antibody is designed to recognize and bind to ATJ2 protein in Arabidopsis thaliana. ATJ2 functions as a molecular chaperone in plant cells and belongs to the DnaJ/Hsp40 family of proteins that play critical roles in protein folding, unfolding, and stress responses. The antibody typically recognizes specific epitopes on the ATJ2 protein with high specificity, allowing researchers to detect its presence and expression levels in various experimental conditions .

What are the primary applications of ATJ2 Antibody in plant research?

The primary applications of ATJ2 antibody in Arabidopsis research include:

• Western Blotting (WB): For detecting ATJ2 protein expression levels in plant tissue extracts

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of ATJ2 protein

These applications make the antibody valuable for studies investigating protein expression, stress responses, and molecular chaperone functions in Arabidopsis research systems .

What sample types can be used with ATJ2 Antibody?

ATJ2 antibody can be used with various sample types from Arabidopsis thaliana, including:

Proper sample preparation is critical for maintaining protein integrity and ensuring successful antibody binding .

What is the optimal protocol for using ATJ2 Antibody in Western blotting?

For optimal Western blotting results with ATJ2 antibody, follow this methodological approach:

• Sample preparation:

  • Homogenize 100-200 mg of plant tissue in 500 μl of extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, protease inhibitor cocktail)

  • Centrifuge at 12,000 × g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• SDS-PAGE and transfer:

  • Load 20-40 μg of protein per lane

  • Separate proteins on 10-12% SDS-PAGE gel

  • Transfer to PVDF or nitrocellulose membrane (100V for 1 hour)

• Antibody incubation:

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

  • Incubate with ATJ2 antibody at 1:1000 dilution overnight at 4°C

  • Wash 3× with TBST

  • Incubate with appropriate secondary antibody (typically anti-rabbit HRP) at 1:5000 for 1 hour

  • Wash 3× with TBST

• Detection:

  • Apply ECL substrate and image using appropriate detection system

  • Expected band size for ATJ2 protein should be verified based on the specific antibody datasheet

This protocol can be adjusted based on specific experimental requirements and antibody performance .

How should ATJ2 Antibody be used in ELISA applications?

For ELISA applications with ATJ2 antibody, follow this methodological framework:

• Plate coating:

  • Dilute capture antibody in coating buffer (100 mM carbonate-bicarbonate buffer, pH 9.6)

  • Add 100 μl per well of 96-well plate

  • Incubate overnight at 4°C

• Sample preparation:

  • Prepare plant extracts in extraction buffer

  • Centrifuge at 12,000 × g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

  • Prepare serial dilutions for standard curve

• Assay procedure:

  • Wash plate 3× with PBST

  • Block with 300 μl of 3% BSA in PBS for 1 hour at room temperature

  • Add 100 μl of samples and standards

  • Incubate for 2 hours at room temperature

  • Wash 5× with PBST

  • Add detection antibody at recommended dilution

  • Incubate for 1 hour at room temperature

  • Wash 5× with PBST

  • Add substrate and measure absorbance

• Data analysis:

  • Generate standard curve

  • Calculate protein concentrations in samples

Optimization of antibody concentrations and incubation times may be required for specific experimental conditions .

What controls should be included when working with ATJ2 Antibody?

Proper experimental controls are essential for validating results with ATJ2 antibody:

Including these controls helps ensure experimental rigor and validates the specificity of observed results .

How can ATJ2 Antibody be used to study stress responses in Arabidopsis?

ATJ2 antibody can be instrumental in studying plant stress responses through these methodological approaches:

• Expression profiling under stress conditions:

  • Subject Arabidopsis plants to various stressors (heat, cold, drought, salt, etc.)

  • Harvest tissues at different time points

  • Extract proteins and perform Western blot analysis with ATJ2 antibody

  • Quantify changes in ATJ2 expression relative to control conditions

  • Correlate expression changes with physiological responses

• Co-immunoprecipitation studies:

  • Use ATJ2 antibody to pull down ATJ2 protein complexes

  • Identify interaction partners under normal and stress conditions

  • Compare interactome changes to elucidate stress-responsive molecular mechanisms

• Subcellular localization studies:

  • Perform cellular fractionation

  • Use ATJ2 antibody to detect protein redistribution under stress

  • Correlate localization changes with functional outcomes

This integrated approach can provide insights into the molecular mechanisms of stress adaptation involving ATJ2 chaperone function .

What methodological considerations are important when studying ATJ2 protein interactions?

When studying ATJ2 protein interactions, consider these critical methodological aspects:

• Co-immunoprecipitation optimization:

  • Lysis buffer composition affects protein complex preservation

  • Use mild detergents (0.1-0.5% NP-40 or Triton X-100)

  • Include protease inhibitors and phosphatase inhibitors if studying phosphorylation

  • Optimize antibody concentration (typically 2-5 μg per mg of total protein)

  • Consider crosslinking approaches for transient interactions

• Proximity ligation assays:

  • Combine ATJ2 antibody with antibodies against putative interaction partners

  • Optimize fixation conditions to preserve protein complexes

  • Include appropriate controls to validate specificity

• Pull-down validation:

  • Confirm interactions using reciprocal immunoprecipitation

  • Validate with orthogonal techniques (e.g., yeast two-hybrid, FRET)

  • Consider size-exclusion chromatography to characterize complex formation

These methodological considerations help ensure robust and reproducible protein interaction studies with ATJ2 antibody .

How can researchers troubleshoot weak or absent signals when using ATJ2 Antibody?

When encountering weak or absent signals with ATJ2 antibody, follow this systematic troubleshooting approach:

• Sample preparation issues:

  • Ensure complete protein extraction from plant tissues

  • Verify protein integrity by Coomassie staining

  • Check for presence of proteases by adding extra protease inhibitors

  • Avoid freeze-thaw cycles of protein samples

• Antibody-related factors:

  • Optimize antibody concentration (try 1:500 to 1:5000 dilutions)

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

  • Test different blocking reagents (BSA vs. milk)

  • Verify antibody storage conditions and expiration

• Detection system optimization:

  • Increase exposure time for Western blots

  • Use more sensitive detection reagents (e.g., enhanced chemiluminescence)

  • For ELISA, optimize substrate incubation time

  • Consider signal amplification methods

• Experimental design modifications:

  • Increase protein loading (50-100 μg per lane)

  • Reduce washing stringency

  • Use fresh transfer buffers and reagents

This methodical approach can help identify and address the specific cause of signal problems .

How should researchers analyze and interpret quantitative data from ATJ2 Antibody experiments?

For robust analysis and interpretation of quantitative data from ATJ2 antibody experiments:

• Western blot quantification:

  • Use digital image acquisition with linear dynamic range

  • Perform densitometry using appropriate software (ImageJ, Image Lab)

  • Normalize to loading controls (actin, tubulin)

  • Include multiple biological replicates (minimum n=3)

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

  • Report fold changes rather than absolute values

• ELISA data analysis:

  • Generate standard curves using known concentrations

  • Ensure R² > 0.98 for reliable quantification

  • Work within the linear range of detection

  • Use appropriate curve-fitting methods (4-parameter logistic regression)

  • Calculate coefficient of variation between technical replicates (<15%)

  • Apply dilution correction factors accurately

• Statistical considerations:

  • Test for normality before applying parametric tests

  • Use appropriate multiple comparison corrections

  • Report both biological and technical variability

  • Consider power analysis to determine sample size requirements

This analytical framework ensures reliable quantitative comparisons and meaningful interpretation of ATJ2 expression data .

How can ATJ2 Antibody be integrated with other techniques for comprehensive protein function studies?

ATJ2 antibody can be integrated with complementary techniques to provide comprehensive insights into protein function:

• Multi-omics integration approaches:

  • Combine ATJ2 antibody-based proteomics with transcriptomics

  • Correlate protein expression (Western blot/ELISA) with mRNA levels (RT-qPCR)

  • Integrate with metabolomic profiling to connect ATJ2 function with metabolic outcomes

  • Develop computational models linking gene expression, protein levels, and physiological responses

• Advanced microscopy applications:

  • Use ATJ2 antibody for super-resolution microscopy to study protein localization

  • Combine with live-cell imaging of fluorescently tagged interacting partners

  • Implement FRET-based approaches to study dynamic interactions in vivo

  • Apply correlative light and electron microscopy for ultrastructural studies

• Functional genomics integration:

  • Compare ATJ2 antibody detection in wild-type vs. mutant lines

  • Correlate protein expression with phenotypic outcomes

  • Combine with CRISPR-Cas9 gene editing to study structure-function relationships

This integrated research approach provides a more complete understanding of ATJ2 protein function in plant biology contexts .

What methodological approaches should be used when contradictory data emerges from ATJ2 Antibody experiments?

When faced with contradictory data from ATJ2 antibody experiments, apply these methodological resolution strategies:

• Antibody validation reassessment:

  • Verify antibody specificity using knockout/knockdown controls

  • Test multiple antibody lots or sources if available

  • Perform epitope mapping to confirm targeting specificity

  • Consider generating new validation data with orthogonal methods

• Technical variation analysis:

  • Systematically evaluate all experimental variables (buffers, incubation times, temperatures)

  • Standardize protocols across different researchers/laboratories

  • Implement blinded experimental design and analysis

  • Develop standard operating procedures (SOPs) for consistent methodology

• Biological context considerations:

  • Evaluate developmental stage differences

  • Consider circadian or diurnal regulation effects

  • Assess environmental condition variations

  • Examine genetic background differences

• Independent validation approaches:

  • Use alternative detection methods (mass spectrometry)

  • Generate transgenic lines with tagged ATJ2 for antibody-independent detection

  • Apply in vitro expression systems to control variables

  • Collaborate with independent laboratories for validation

This systematic approach helps resolve contradictions and advances scientific understanding through methodological rigor .

How does ATJ2 Antibody performance compare to other molecular tools for studying chaperone proteins in plants?

A comparative analysis of ATJ2 antibody against other molecular tools reveals distinct advantages and limitations:

This comparative analysis helps researchers select the most appropriate tool based on specific experimental questions and available resources .

What experimental design considerations are critical when using ATJ2 Antibody for developmental studies in Arabidopsis?

When designing experiments with ATJ2 antibody for developmental studies, implement these methodological considerations:

• Developmental stage sampling strategy:

  • Clearly define developmental stages using standardized metrics

  • Sample at regular intervals throughout development

  • Consider tissue-specific expression patterns

  • Use synchronized plant populations to reduce variability

• Experimental controls expansion:

  • Include developmental time-course controls

  • Compare multiple tissue types at each stage

  • Utilize tissue-specific promoter lines as references

  • Implement hormone-response mutants as functional controls

• Quantification approaches:

  • Normalize protein expression to stage-specific reference proteins

  • Implement ratiometric analysis against constitutive controls

  • Consider relative vs. absolute quantification methods

  • Develop tissue-specific protein extraction protocols

• Validation framework:

  • Confirm antibody specificity across developmental stages

  • Verify with transgenic reporter lines when possible

  • Correlate protein patterns with known developmental markers

  • Implement genetic complementation to confirm functionality

This comprehensive experimental design framework ensures robust developmental studies using ATJ2 antibody in plant research contexts .