ATJ72 Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
What is ATJ72 and what are its primary functional characteristics?
ATJ72 (AT2G41000) is a chaperone protein belonging to the DnaJ superfamily in Arabidopsis thaliana. It functions as a molecular chaperone that assists in protein folding, unfolding, transport, and degradation processes. As a member of the DnaJ family, it contains the characteristic J-domain that mediates interactions with Hsp70 proteins to regulate ATPase activity . The protein is encoded by genes identified as ATJ72, AT2G41000, C72, LCR51, T3K9.23, and T3K9_23, reflecting various annotations across different databases and research contexts .
When studying ATJ72, researchers should note that its chaperone activity is often context-dependent, requiring careful experimental design that accounts for physiological conditions relevant to plant stress responses. Methodologically, initial characterization typically involves recombinant protein expression followed by functional assays measuring chaperone activity under varying conditions of temperature, pH, and co-factor concentration.
What experimental applications are suitable for ATJ72 antibodies?
ATJ72 antibodies have demonstrated utility in several key experimental applications:
| Application | Sample Preparation | Detection Method | Key Considerations |
|---|---|---|---|
| Western Blot | Protein extraction from plant tissue, SDS-PAGE separation | Rabbit polyclonal anti-ATJ72 antibody | Recommended dilution: 1:1000-1:5000; Blocking: 5% non-fat milk |
| ELISA | Protein extraction in non-denaturing buffer | Direct or sandwich ELISA with anti-ATJ72 antibody | Sensitivity threshold: ~10ng/mL; Standard curve range: 10-1000ng/mL |
| Immunoprecipitation | Native protein extraction | Anti-ATJ72 coupled to protein A/G beads | Gentle elution to maintain interacting protein complexes |
| Immunofluorescence | Fixed plant tissue sections or protoplasts | Fluorophore-conjugated secondary antibody | Permeabilization critical for accessing intracellular targets |
When designing experiments, researchers should validate antibody specificity using appropriate controls including pre-immune serum and blocking peptides. For optimal results in Western blot applications, ensure proper antigen identification through molecular weight markers and positive controls .
How should researchers evaluate ATJ72 antibody specificity?
Evaluating antibody specificity is crucial for reliable experimental outcomes. For ATJ72 antibodies specifically:
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Cross-reactivity assessment: Test against recombinant ATJ72 proteins and related DnaJ family members to confirm specificity within the chaperone family.
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Knockout/knockdown validation: Compare antibody performance in wild-type versus ATJ72 knockout/knockdown plant lines.
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Epitope mapping: Determine which regions of ATJ72 the antibody recognizes using peptide arrays or truncated recombinant proteins.
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Pre-absorption controls: Pre-incubate antibody with purified antigen to confirm signal reduction in subsequent assays.
Methodologically, researchers should implement a rigorous validation workflow that includes parallel testing with multiple antibody lots and detection techniques. Document batch variations and optimize working concentrations for each experimental system individually, as antibody performance can vary significantly between applications .
What strategies can address epitope accessibility challenges when targeting ATJ72 in different cellular compartments?
ATJ72 localization and epitope accessibility can vary depending on cellular conditions and protein interactions. Advanced researchers should consider:
| Cellular Compartment | Accessibility Challenge | Technical Solution | Validation Method |
|---|---|---|---|
| Cytosolic | Transient interactions with Hsp70 | Crosslinking prior to extraction | Co-immunoprecipitation |
| Membrane-associated | Masked epitopes | Membrane fractionation | Protease protection assays |
| Stress granules | Aggregation complexes | Detergent optimization | Immunofluorescence colocalization |
| Nuclear | Low abundance | Nuclear enrichment protocols | Subcellular fractionation controls |
When developing methodological approaches:
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Fixation optimization: Test multiple fixation methods (paraformaldehyde, methanol, acetone) to preserve epitope structure while allowing antibody access.
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Antigen retrieval: For fixed tissue sections, evaluate heat-induced or enzymatic antigen retrieval methods to expose masked epitopes.
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Permeabilization gradient: Establish optimal detergent concentration and exposure time to maintain structural integrity while allowing antibody penetration.
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Live-cell approaches: Consider developing smaller antibody fragments (Fabs, scFvs, or nanobodies) that may have better access to sterically hindered epitopes.
Researchers should implement systematic optimization of these parameters, documenting conditions that maximize signal-to-noise ratio while maintaining biological relevance of the detected interactions .
What are the most effective methods for troubleshooting non-specific binding issues with ATJ72 antibodies?
Non-specific binding represents a common challenge when working with antibodies against plant proteins like ATJ72. Advanced troubleshooting approaches include:
| Problem | Potential Cause | Solution Strategy | Validation Method |
|---|---|---|---|
| High background in Western blots | Insufficient blocking | Optimize blocking agent (BSA vs. milk) and concentration | Signal-to-noise ratio quantification |
| Cross-reactivity with related proteins | Conserved epitopes | Affinity purification against specific regions | Pre-absorption with related proteins |
| Variable results between experiments | Antibody degradation | Aliquot storage and freeze-thaw monitoring | Control blot with standard sample |
| Non-specific bands | Post-translational modifications | Include phosphatase/deglycosylase treatments | Mass spectrometry of bands |
Methodological approach for systematic troubleshooting:
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Epitope mapping: Identify precisely which region of ATJ72 the antibody recognizes to predict potential cross-reactivity with related DnaJ proteins.
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Buffer optimization: Systematically test different detergents, salt concentrations, and pH conditions to minimize non-specific interactions while maintaining specific binding.
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Alternative detection systems: Compare performance across different secondary antibodies and detection methods (chemiluminescence, fluorescence, colorimetric).
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Competitive assays: Perform titration experiments with purified recombinant ATJ72 to demonstrate signal reduction, confirming specificity.
Researchers should document all optimization parameters in a structured format, enabling reproducible protocols and facilitating troubleshooting when transitioning methods between different experimental systems .
