DJC76 Antibody

What is DJC76 protein and what cellular functions does it perform?

DJC76 (also known as AtDjC17 or AtJ17) is a chaperone protein belonging to the DnaJ family, located in the chloroplast of Arabidopsis thaliana. Evidence indicates that it collaborates with the HSC70 chaperone to facilitate protein folding and prevent aggregation, particularly under chloroplast salt stress conditions. Additionally, it plays a crucial role in root development and the precise determination of cell fate during root hair development. As a member of the DnaJ protein family, it likely functions as a co-chaperone that regulates HSP70 activity by stimulating its ATPase activity, thereby assisting in protein folding, transport, and degradation processes within plant cells.

What are the optimal storage conditions for DJC76 antibody?

The DJC76 antibody should be stored at -20°C or -80°C immediately upon receipt to maintain its stability and activity. Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of antibody functionality . For short-term storage during experimental work, the antibody can be kept at 4°C for up to one week. The antibody is supplied in a storage buffer containing 0.03% Proclin 300 (as a preservative) and 50% glycerol in 0.01M PBS at pH 7.4, which helps maintain stability during freeze-thaw cycles .

What applications has the DJC76 antibody been validated for?

The DJC76 antibody has been specifically validated for ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot (WB) applications . These techniques are commonly used to detect and quantify the DJC76 protein in plant tissue samples. The antibody can be used to study protein expression patterns across different tissues or under various experimental conditions. While not explicitly validated for other applications, researchers might explore its utility in immunohistochemistry or immunofluorescence to study the subcellular localization of DJC76, particularly given its known chloroplastic localization.

What is the species reactivity of DJC76 antibody?

The DJC76 antibody has been specifically raised against and validated for Arabidopsis thaliana (mouse-ear cress) . Cross-reactivity with other plant species has not been explicitly reported in the provided information, although proteins with high sequence homology in related species might potentially be recognized. Researchers working with other plant species would need to perform their own validation tests to confirm cross-reactivity before using this antibody in their studies.

What is the recommended protocol for Western blotting with DJC76 antibody?

Sample Preparation:

• Extract total protein from Arabidopsis tissue using a standard extraction buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA with protease inhibitors)

• Quantify protein concentration using Bradford or BCA assay

• Prepare samples in Laemmli buffer and denature at 95°C for 5 minutes

SDS-PAGE and Transfer:

• Load 10-30 μg of protein per lane on a 10-12% SDS-PAGE gel

• Separate proteins by electrophoresis at 100-120V

• Transfer to a PVDF or nitrocellulose membrane (25V for 1.5 hours or 100V for 1 hour)

Immunoblotting:

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

• Incubate with DJC76 primary antibody at 1:1000 to 1:2000 dilution in blocking buffer overnight at 4°C

• Wash 3× with TBST, 5 minutes each

• Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature

• Wash 3× with TBST, 5 minutes each

• Develop using ECL substrate and detect signal using a chemiluminescence imaging system

This protocol is based on standard Western blotting procedures using polyclonal antibodies similar to DJC76 antibody, which is a rabbit polyclonal antibody . Optimization might be needed for specific experimental conditions.

How can I optimize ELISA protocols using DJC76 antibody?

Indirect ELISA Protocol:

• Coating:

  • Coat microplate wells with antigen (recombinant DJC76 protein or plant extract) at 1-10 μg/ml in carbonate buffer (pH 9.6)

  • Incubate overnight at 4°C

• Blocking:

  • Block with 1-3% BSA in PBS for 1-2 hours at room temperature

• Primary Antibody:

  • Prepare a dilution series of DJC76 antibody (1:500 to 1:5000) in blocking buffer

  • Add to wells and incubate for 2 hours at room temperature

• Secondary Antibody:

  • Add HRP-conjugated anti-rabbit IgG (1:5000) in blocking buffer

  • Incubate for 1 hour at room temperature

• Detection:

  • Add TMB substrate and incubate for 15-30 minutes

  • Stop reaction with 2N H₂SO₄

  • Measure absorbance at 450 nm

Optimization Tips:

• Perform checkerboard titration to determine optimal antibody concentration

• Include controls: no primary antibody, no antigen, and a positive control if available

• Compare different blocking agents (BSA, casein, non-fat dry milk) to reduce background

• Test various incubation times and temperatures for optimal signal-to-noise ratio

This approach is based on standard ELISA protocols for polyclonal antibodies, as DJC76 antibody has been validated for ELISA applications .

What controls should be included when working with DJC76 antibody in immunoassays?

Essential Controls for DJC76 Antibody Experiments:

• Positive Control:

  • Arabidopsis thaliana root tissue lysate (where DJC76 is expressed specifically in the stele)

  • Recombinant DJC76 protein (if available)

• Negative Controls:

  • No primary antibody control (secondary antibody only)

  • Pre-immune serum control (if available)

  • Tissue from DJC76 knockout plants (if available)

  • Non-target tissue where DJC76 expression is minimal or absent

• Specificity Controls:

  • Blocking peptide competition assay: pre-incubate antibody with excess immunizing peptide to confirm signal specificity

  • Validate bands or signals using recombinant DJC76 protein as a molecular weight reference

• Loading Controls for Western Blots:

  • Housekeeping proteins (β-actin, GAPDH, or α-tubulin)

  • Total protein staining (Ponceau S, SYPRO Ruby)

• Validation Method:

  • If possible, validate key findings with a second antibody targeting a different epitope of DJC76 or using orthogonal methods (e.g., mass spectrometry, RT-PCR)

Including these controls helps establish antibody specificity and experimental validity, which is critical for scientific rigor in immunoassay-based experiments.

How can the DJC76 antibody be used to study plant stress responses?

Experimental Approach:

• Stress Treatment Design:

  • Expose Arabidopsis plants to various stressors (salt, drought, heat, cold, oxidative stress)

  • Include time-course sampling (0, 1, 3, 6, 12, 24, 48 hours)

  • Maintain appropriate controls for each stress condition

• Protein Expression Analysis:

  • Extract proteins from stressed and control plants

  • Perform Western blot analysis using DJC76 antibody to quantify expression changes

  • Normalize to loading controls and calculate fold changes

• Subcellular Localization:

  • Perform subcellular fractionation to isolate chloroplasts

  • Compare DJC76 levels in chloroplastic versus non-chloroplastic fractions

  • Use confocal microscopy with fluorescently-tagged secondary antibodies to visualize potential stress-induced relocalization

• Protein Interaction Studies:

  • Use co-immunoprecipitation with DJC76 antibody to identify stress-responsive protein interactions

  • Compare interactome under normal versus stress conditions

  • Focus on HSC70 interactions, as DJC76 is known to collaborate with HSC70 chaperones

• Data Analysis:

  • Create expression profiles across stress conditions and timepoints

  • Perform statistical analysis to identify significant changes

  • Correlate DJC76 expression patterns with physiological stress markers

This approach leverages the DJC76 antibody to investigate how this chloroplastic chaperone responds to environmental stresses, potentially revealing its role in stress adaptation mechanisms in plants.

What are effective strategies for studying DJC76 interactions with HSC70 chaperones?

Methodological Approaches:

• Co-Immunoprecipitation (Co-IP):

  • Lyse plant tissues under non-denaturing conditions

  • Immobilize DJC76 antibody on protein A/G beads

  • Incubate with plant lysate to capture DJC76 and associated proteins

  • Elute complexes and analyze by Western blot using HSC70-specific antibodies

  • Perform reciprocal Co-IP with HSC70 antibodies and detect DJC76

• Proximity Ligation Assay (PLA):

  • Fix and permeabilize plant cells or tissues

  • Incubate with both DJC76 and HSC70 primary antibodies

  • Apply species-specific PLA probes

  • Perform ligation and amplification per PLA protocol

  • Visualize interaction signals via fluorescence microscopy

• Bimolecular Fluorescence Complementation (BiFC):

  • Generate fusion constructs of DJC76 and HSC70 with split fluorescent protein fragments

  • Transform plants or protoplasts with both constructs

  • Analyze reconstituted fluorescence as indication of protein interaction

  • Quantify fluorescence intensity under various conditions

• Pull-down Assays:

  • Express recombinant tagged DJC76 protein

  • Immobilize on affinity resin

  • Incubate with plant lysates containing HSC70

  • Wash and elute bound proteins

  • Detect interactions via Western blot

• Microscale Thermophoresis (MST):

  • Fluorescently label purified DJC76 or HSC70

  • Titrate with increasing concentrations of the unlabeled partner

  • Measure thermophoretic mobility changes

  • Calculate binding affinities (Kd values)

These approaches provide complementary data on DJC76-HSC70 interactions, with each method offering different advantages in terms of sensitivity, specificity, and ability to detect transient or weak interactions.

How can researchers investigate the role of DJC76 in root development using the antibody?

Comprehensive Experimental Strategy:

• Developmental Expression Profiling:

  • Collect root samples at different developmental stages

  • Section roots to analyze cell-type specific expression

  • Perform immunohistochemistry with DJC76 antibody

  • Quantify signal intensity across developmental zones and cell types

• Co-localization Studies:

  • Perform double immunolabeling with DJC76 antibody and markers for:

    • Cell division (cyclin markers)

    • Root hair specification (RHD6, RSL4)

    • Vascular development markers

  • Analyze co-localization patterns using confocal microscopy

• Genetic Approaches:

  • Compare DJC76 protein levels in wild-type plants versus:

    • DJC76 overexpression lines

    • DJC76 knockout/knockdown mutants

    • Root development mutants (e.g., scr, shr, wer)

  • Validate antibody specificity using knockout lines

  • Quantify protein expression changes via Western blot

• Physiological Treatments:

  • Apply hormones that affect root development (auxin, cytokinin, ethylene)

  • Monitor DJC76 protein abundance changes via immunoblotting

  • Correlate with phenotypic alterations in root architecture

• Protein Complex Analysis:

  • Use the DJC76 antibody for immunoprecipitation from root tissues

  • Identify root-specific interaction partners via mass spectrometry

  • Validate key interactions with complementary methods

• Data Integration:

  • Create spatiotemporal maps of DJC76 expression in roots

  • Correlate protein abundance with root developmental processes

  • Develop models for DJC76 function in root hair determination

This multifaceted approach capitalizes on the tissue specificity of DJC76, which is expressed exclusively in the stele of roots, to elucidate its precise role in root development.

How can researchers troubleshoot weak or absent signals when using DJC76 antibody?

Systematic Troubleshooting Guide:

Special Considerations for DJC76:

• Remember that DJC76 is expressed specifically in roots, exclusively in the stele tissue

• Expression levels might be low in whole plant or mixed tissue samples

• Use enriched root samples or increase total protein loading

• Consider chloroplast isolation to enrich for DJC76 protein

These systematic approaches should help identify and resolve common issues when working with the DJC76 antibody in various applications.

How can researchers validate the specificity of DJC76 antibody to ensure accurate experimental results?

Comprehensive Antibody Validation Strategy:

• Genetic Validation:

  • Compare signal between wild-type and DJC76 knockout/knockdown plants

  • Expected result: Significant reduction or absence of signal in knockout samples

  • This provides the strongest validation of antibody specificity

• Molecular Weight Confirmation:

  • Compare detected band with theoretical molecular weight of DJC76

  • DnaJ family proteins typically show characteristic molecular weight patterns

  • Use size markers and recombinant protein controls when available

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide or recombinant DJC76

  • Apply to parallel samples in Western blot or immunohistochemistry

  • Expected result: Signal should be blocked or significantly reduced in the peptide-blocked sample

• Tissue-Specific Expression Pattern:

  • Validate that strongest signals appear in root stele tissue

  • Compare with known expression patterns from transcriptomic data

  • Expected result: Signal intensity should correlate with known tissue-specific expression

• Orthogonal Method Validation:

  • Compare protein detection with mRNA expression (RT-PCR, RNA-seq)

  • Correlate protein levels with functional assays or phenotypes

  • Use mass spectrometry to confirm protein identity in immunoprecipitated samples

• Cross-Reactivity Assessment:

  • Test antibody against recombinant proteins from related DnaJ family members

  • Expected result: Minimal cross-reactivity with other family members

• Reproducibility Testing:

  • Repeat key experiments with different antibody lots

  • Compare results across different experimental conditions

Documenting these validation steps thoroughly increases confidence in results and should be included in publications using this antibody.

What are the potential pitfalls in interpreting immunolocalization data with DJC76 antibody?

Critical Considerations for Accurate Interpretation:

These considerations help researchers critically evaluate immunolocalization results with DJC76 antibody and avoid common misinterpretations.

How might researchers apply DJC76 antibody in studying chloroplast stress response mechanisms?

Innovative Research Applications:

• Stress-Induced Protein Aggregation Studies:

  • Use the DJC76 antibody to monitor co-localization with protein aggregates during heat or salt stress

  • Analyze the temporal dynamics of DJC76 recruitment to stress granules

  • Correlate DJC76 levels with chloroplast protein homeostasis markers

• Chloroplast Unfolded Protein Response Investigation:

  • Monitor DJC76 expression and localization during chloroplast UPR

  • Use the antibody to track changes in DJC76-HSC70 interactions under stress

  • Compare wild-type vs. UPR mutant responses

• Thylakoid Membrane Protection:

  • Investigate potential associations between DJC76 and thylakoid membrane proteins

  • Use immunogold electron microscopy with DJC76 antibody to precisely localize the protein within chloroplast subcompartments

  • Test if DJC76 protects photosynthetic complexes during high light stress

• Organellar Communication:

  • Study whether DJC76 participates in retrograde signaling from chloroplast to nucleus

  • Analyze nuclear localization of transcription factors that might be regulated by DJC76-mediated pathways

  • Use the antibody to test if DJC76 relocates during severe stress conditions

• Methodological Approach:

  • Combine DJC76 immunoprecipitation with proteomics to identify stress-specific interactors

  • Use proximity labeling techniques with DJC76 antibody to capture transient interactions

  • Develop DJC76 activity assays to measure chaperone function under stress conditions

This research direction would leverage the unique properties of DJC76 as a chloroplastic DnaJ protein to gain new insights into how plants maintain chloroplast proteostasis during environmental stress.

What experimental approaches could elucidate the mechanism of DJC76 in determining cell fate during root hair development?

Cutting-Edge Experimental Design:

• Single-Cell Protein Profiling:

  • Use the DJC76 antibody in fluorescence-activated cell sorting (FACS) of root protoplasts

  • Analyze cell-type specific expression across root hair vs. non-root hair cell lineages

  • Correlate with transcriptomic profiles from the same cell populations

• Temporal Dynamics Analysis:

  • Perform time-course immunostaining during root hair initiation and development

  • Track DJC76 localization changes during trichoblast specification

  • Use time-lapse imaging with fluorescently-labeled antibody in living tissue

• Protein-Chromatin Interaction Studies:

  • Perform ChIP-seq using DJC76 antibody to identify potential gene targets

  • Focus on genes known to regulate root hair development

  • Validate interactions with reporter gene assays

• Mechanistic Pathway Analysis:

  • Use the antibody to immunoprecipitate DJC76 from different root cell types

  • Identify cell-type specific interaction partners via mass spectrometry

  • Map DJC76 into known root hair specification pathways

• Perturbation Experiments:

  • Create inducible DJC76 expression systems

  • Use the antibody to confirm protein expression timing

  • Observe phenotypic consequences on root hair patterning

• Super-Resolution Visualization:

  • Apply techniques like STORM or PALM using DJC76 antibody

  • Achieve nanometer-scale resolution of DJC76 localization

  • Compare distribution in trichoblast vs. atrichoblast cell files

This multifaceted approach would help determine whether DJC76 influences root hair development through direct transcriptional regulation, protein folding assistance for developmental regulators, or other novel mechanisms.

What are the considerations for using DJC76 antibody in cross-species comparative studies?

Critical Factors for Comparative Research:

• Sequence Homology Assessment:

  • Perform bioinformatic analysis to identify DJC76 homologs across plant species

  • Generate sequence alignments to predict cross-reactivity potential

  • Focus on conservation of the immunogen region used to generate the antibody

• Epitope Conservation Analysis:

  • Use epitope prediction tools to evaluate conservation of antibody binding sites

  • Examine 3D structural conservation when available

  • Consider testing with synthetic peptides from different species

• Validation Strategy:

  • Perform controlled Western blot tests on each species of interest

  • Include positive controls (Arabidopsis) alongside test species

  • Use genetic knockouts or knockdowns in non-Arabidopsis species when available

  • Consider using heterologous expression systems to confirm specificity

• Technical Optimization Table:

  Species Group	Expected Homology	Recommended Approach
  Brassicaceae family	High	Standard protocols with minor dilution adjustments
  Other dicots	Moderate	Increased antibody concentration, extended incubation
  Monocots	Low to moderate	Epitope retrieval techniques, higher antibody concentration
  Non-vascular plants	Low	Preliminary testing essential, may require new antibody

• Data Interpretation Guidelines:

  • Account for evolutionary divergence in molecular weight and post-translational modifications

  • Consider differential expression patterns across species

  • Be cautious about functional interpretations without supporting evidence

  • Clearly document limitations in cross-species applications

• Alternative Approaches:

  • For distantly related species, consider generating species-specific antibodies

  • Use orthogonal methods (mass spectrometry, transcriptomics) to support antibody results

  • Apply genetic tagging approaches in non-model species

These considerations provide a framework for expanding DJC76 research beyond Arabidopsis thaliana, while maintaining scientific rigor and acknowledging potential limitations.