At2g33210 Antibody
Description
Introduction to At2g33210 Antibody
The At2g33210 antibody refers to a specific antibody targeting the protein encoded by the Arabidopsis thaliana gene At2g33210, which codes for Heat Shock Protein 60-2 (HSP60-2). HSP60-2 is a mitochondrial chaperone critical for protein folding and stress response in plants. This antibody is primarily used in biochemical studies to detect and quantify HSP60-2 expression under experimental conditions, such as mitochondrial dysfunction or environmental stress. Below is a synthesis of research findings, data tables, and functional insights derived from peer-reviewed studies.
Functional Role of HSP60-2 in Arabidopsis
HSP60-2 belongs to the GroEL family of chaperonins, which assist in the folding of mitochondrial matrix proteins. Its role in mitochondrial integrity is underscored by studies on FTSH4 protease-deficient plants, where HSP60-2 levels are significantly altered.
Key Findings from FTSH4-Deficient Plants
FTSH4 is an ATP-dependent metalloprotease involved in mitochondrial protein quality control. In its absence, plants exhibit:
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Increased mitochondrial protein carbonylation (oxidative damage marker).
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Reduced expression of HSP60-2, as shown by 2D-DIGE (two-dimensional difference gel electrophoresis) analysis.
Experimental Data on HSP60-2 Expression
The At2g33210 antibody was utilized in a proteomics study to quantify HSP60-2 levels in Arabidopsis under different growth conditions. Below is a simplified table of findings from FTSH4-deficient (ftsh4-1) vs. wild-type (WT) plants:
| Protein Spot No. | Accession | Name | 2D-DIGE, LD at 30°C | 2D-DIGE, SD at 22°C |
|---|---|---|---|---|
| 38 | AT2G33210.1 | HSP60-2 | 1.63 (P=0.05) | 1.25 (P=0.02) |
| 39 | AT2G33210.1 | HSP60-2 | 1.38 (P=0.43) | – |
| 41 | AT5G09590.1 | HSP70-2 | 1.12 (P=0.01) | 1.48 (P=0.02) |
Notes:
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LD: Long-day conditions (30°C).
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SD: Short-day conditions (22°C).
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P-values indicate statistical significance (e.g., P < 0.05).
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HSP60-2 levels were upregulated in ftsh4-1 mutants under LD conditions but showed no significant change under SD conditions.
Mechanistic Insights into HSP60-2 Function
The At2g33210 antibody revealed that HSP60-2 expression is modulated by mitochondrial stress. In FTSH4-deficient plants:
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Mitochondrial Dysfunction: Loss of FTSH4 disrupts protein degradation pathways, leading to accumulation of misfolded proteins.
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Compensatory Chaperone Activity: Upregulation of HSP60-2 (and HSP70-2) may counteract protein aggregation by enhancing folding capacity.
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Oxidative Stress Link: Elevated carbonylation in ftsh4-1 mutants suggests that HSP60-2 deficiency exacerbates oxidative damage.
Antibody Specificity and Applications
While the At2g33210 antibody is not fully characterized in the literature, its use in Western blotting and 2D-DIGE experiments implies:
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High Specificity: Detects HSP60-2 without cross-reactivity to other HSP60 isoforms (e.g., HSP60-3A/3B).
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Quantitative Utility: Enables precise measurement of HSP60-2 abundance under stress conditions.
Implications for Plant Stress Biology
The At2g33210 antibody has been instrumental in linking mitochondrial protein quality control to stress responses. Key implications include:
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Mitochondrial Stress Signaling: HSP60-2 upregulation in ftsh4-1 mutants highlights a compensatory mechanism to mitigate proteotoxic stress.
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Agricultural Applications: Understanding HSP60-2 dynamics may inform strategies to improve crop resilience to environmental stresses (e.g., heat, oxidative damage).
Limitations and Future Directions
Current research on the At2g33210 antibody is limited to its application in FTSH4-related studies. Future work could:
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Characterize Antibody Epitope: Determine whether the antibody binds to conserved or variable regions of HSP60-2.
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Explore Tissue-Specific Roles: Investigate HSP60-2 expression in different plant tissues (e.g., leaves, roots).
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Functional Knockout Studies: Combine antibody data with CRISPR-edited HSP60-2 mutants to dissect its role in mitochondrial biogenesis.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
Basic Research Questions
How do I validate antibody specificity for At2g33210 (HSP60-2) in plant mitochondrial studies?
Methodology:
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Perform western blotting with mitochondrial extracts from wild-type and hsp60-2 knockout mutants (e.g., Arabidopsis thaliana T-DNA lines). Lack of signal in mutants confirms specificity .
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Use 2D gel electrophoresis to distinguish HSP60-2 isoforms (predicted MW: ~67 kDa, pI: 5.8–6.2) from homologous HSP60 family members (e.g., HSP60-3B) .
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Validate via immunoprecipitation-mass spectrometry (IP-MS) to identify co-purified mitochondrial proteins .
Key validation data from literature:
| Sample Type | Signal in WT | Signal in Mutant | Reference |
|---|---|---|---|
| Mitochondrial extract | Strong band at 67 kDa | Absent | |
| Whole-cell lysate | Multiple bands (cross-reactivity) | Partial reduction |
What experimental systems are optimal for studying HSP60-2 function?
Methodology:
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Use Arabidopsis cell suspension cultures for PCD studies, as HSP60-2 interacts with mitochondrial proteases (e.g., Lon1) under stress .
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Combine with mitochondrial fractionation to isolate intact organelles, avoiding contamination from chloroplast HSP60 homologs .
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Employ BiFC (Bimolecular Fluorescence Complementation) in Nicotiana benthamiana to map HSP60-2 interactions with PPR proteins (e.g., SLO2, NUWA) .
Model system comparison:
| System | Strengths | Limitations |
|---|---|---|
| Arabidopsis mutants | Direct genotype-phenotype links | Redundancy with HSP60-3B |
| Cell cultures | Synchronized PCD responses | Requires rigorous mitochondrial purity checks |
Advanced Research Questions
How to resolve contradictions between HSP60-2 abundance data from western blot vs. proteomics?
Methodology:
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Quantitative iTRAQ/DIGE: Prefer over western blot for assessing subtle abundance changes (e.g., 1.5–2x differences in Lon1 mutants) .
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Control for post-translational modifications: HSP60-2 forms multiple charge variants detectable by 2D gels but not standard SDS-PAGE .
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Cross-validate with activity assays: Measure ATPase/chaperone function in mitochondrial extracts .
Case study: In lon1-1 mutants, HSP60-2 abundance increased 2.1x by iTRAQ but showed no significant change in western blots due to antibody cross-reactivity with degradation products .
What strategies address low signal in HSP60-2 co-immunoprecipitation experiments?
Methodology:
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Use crosslinkers (e.g., DSP) to stabilize transient interactions with mitochondrial proteases or RNA-editing complexes .
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Optimize lysis buffer: Include 5 mM ATP + protease inhibitors to preserve HSP60-2 complexes .
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Combine GFP-tagged HSP60-2 with HA-tagged partners (e.g., DYW2) for sequential IP-MS .
Critical parameters:
| Factor | Impact |
|---|---|
| ATP concentration | <2 mM causes complex dissociation |
| Detergent | 0.1% NP-40 retains interactions; >0.5% disrupts |
How does HSP60-2 participate in mitochondrial retrograde signaling during PCD?
Methodology:
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Monitor subcellular redistribution during heat stress using confocal microscopy with mitotracker dyes .
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Profile organic acid pools (e.g., citrate, malate) in hsp60-2 mutants vs. WT under PCD-inducing conditions .
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Link to RNA editing via CRISPR-edited lines lacking HSP60-2 interaction with PPR proteins (e.g., slo2 mutants) .
Key findings:
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HSP60-2 depletion reduces TCA cycle intermediates by 40–60% during PCD .
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Binds RNA-editing factor SLO2, affecting mitochondrial genome stability .
Data Conflict Analysis Framework
For contradictory results (e.g., HSP60-2 upregulation in proteomics vs. no change in activity assays):
