At1g54220 Antibody
Description
Definition and Biological Context
The At1g54220 antibody is designed to detect the E2 subunit of the mitochondrial pyruvate dehydrogenase complex (PDC) in Arabidopsis thaliana. This subunit plays a critical role in catalyzing the oxidative decarboxylation of pyruvate during cellular respiration .
Key Features:
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Target Protein: Pyruvate dehydrogenase E2 subunit (PDC-E2).
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Gene Function: Part of the PDC, which links glycolysis to the citric acid cycle.
Research Applications
The antibody is utilized in plant biochemistry and mitochondrial studies:
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Mitochondrial Proteomics: Identification of PDC-E2 in Arabidopsis mitochondrial extracts via 2D gel electrophoresis and immunoblotting .
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Enzyme Complex Analysis: Mapping subunit composition and post-translational modifications in PDC .
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Cross-Species Studies: Comparative analysis of PDC-E2 homologs in rice (Oryza sativa) and pea (Pisum sativum) .
Key Research Findings
Data from proteomic studies highlight the antibody’s utility in identifying mitochondrial proteins:
Table 1: Identification of At1g54220 in Arabidopsis Mitochondria
| Spot No. | Accession No. | Description | MM Gel (kDa) | Coverage (%) | MOWSE Score |
|---|---|---|---|---|---|
| A3 | At1g54220 | Pyruvate dehydrogenase E2 | 63 | 26 | 636 |
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Coverage: 26% peptide coverage confirms specificity.
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MOWSE Score: A high score (636) validates robust antigen-antibody interaction.
Technical Validation
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Specificity: Recognizes the ~63 kDa PDC-E2 band in Arabidopsis mitochondrial extracts, with no cross-reactivity reported in rice or pea homologs .
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Functional Assays: Used to measure PDC activity in mitochondrial lysates, confirming its role in energy metabolism .
Significance in Plant Biochemistry
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The antibody has enabled insights into diurnal changes in mitochondrial enzyme activity, linking PDC-E2 expression to metabolic rhythms in plants .
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It aids in studying post-translational modifications (e.g., lipoylation) critical for PDC function .
Limitations and Future Directions
Product Specs
Components: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
The At1g54220 antibody is a specialized research tool primarily used in plant molecular biology studies, particularly in Arabidopsis thaliana research. Below are structured FAQs reflecting common technical challenges and methodological considerations in academic research contexts:
Advanced Experimental Design
How to resolve contradictory results between Western blot and GFP-tagged protein localization studies?
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Methodological approach:
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Confirm antibody cross-reactivity via peptide blocking assays
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Compare temporal expression patterns (antibody vs. fluorescent tag)
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Validate using independent transgenic lines
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Perform subcellular fractionation with compartment-specific markers
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What controls are essential when studying protein-protein interactions using At1g54220 antibody in co-IP experiments?
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Negative controls:
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Untagged bait protein lysate
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Empty vector-transfected plant extracts
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Positive controls:
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Known interacting partners from literature
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Bimolecular fluorescence complementation (BiFC) validation
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Technical Optimization
How to improve signal-to-noise ratio in electron microscopy applications?
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Pre-embedding labeling with:
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0.25% glutaraldehyde + 4% PFA fixation
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Triton X-100 permeabilization (0.1-0.3% concentration gradient)
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Data Interpretation Challenges
How to distinguish between true biological variation and technical artifacts in quantitative assays?
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Implement a standardized validation matrix:
Parameter Acceptance Criteria Inter-assay CV <15% across 3 independent runs Intra-sample CV <10% for triplicate wells Spike recovery 85-115% of expected values
