FUM1 Antibody
Description
Introduction to FUM1 Antibody
The FUM1 Antibody is a polyclonal rabbit antibody designed to detect fumarase 1 (FUM1), a mitochondrial enzyme critical for the tricarboxylic acid (TCA) cycle in Arabidopsis thaliana. It is also cross-reactive with the cytosolic isoform FUM2, enabling simultaneous detection of both mitochondrial and cytosolic fumarase in plants .
Key Features
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Dual Specificity: Detects both mitochondrial (FUM1) and cytosolic (FUM2) isoforms due to conserved epitopes .
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Epitope Recognition: Targets peptides derived from the N-terminal regions of FUM1 and FUM2, which include mitochondrial targeting sequences .
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Stability: Stable at -20°C for 1 year or 2–8°C for 1 month; avoid repeated freeze-thaw cycles .
Western Blot Protocols
The antibody is validated for western blotting to study fumarase localization and expression in Arabidopsis:
Detection of FUM1 and FUM2 in Arabidopsis
The antibody has been used to:
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Identify FUM2 mutants: In fum2-1 mutants, the antibody detects residual FUM1 (~60 kDa) due to mitochondrial retention, while FUM2 is absent .
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Track GFP-FUM2 complementation: Confirms reexpression of GFP-tagged FUM2 in fum2-1 mutants .
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Compare wild-type and mutant strains: Distinguishes FUM1/FUM2 expression profiles in fum2-1 and fum2-2 mutants .
Table 1: Western Blot Results in Arabidopsis Strains
Mechanistic Insights from Yeast Studies
While not directly tested with the FUM1 Antibody, yeast studies reveal:
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Single precursor processing: FUM1 encodes a single precursor translocated into mitochondria, with excess folding into the cytosol .
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Cross-reactivity potential: The antibody’s epitope alignment with yeast FUM1 could enable detection in other eukaryotes, though unverified .
Considerations for Use and Handling
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Reactivity: Primarily validated for Arabidopsis; cross-reactivity with other species (e.g., Ananas comosus) requires experimental confirmation .
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Optimal Conditions: Avoid prolonged storage at 2–8°C; use fresh dilutions for each experiment .
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Controls: Include secondary antibody-only lanes to rule out non-specific binding .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research indicates that the signal transduction mechanism operates through Ca(2+) activation of the gene encoding the transcription factor PIF3. PIF3 binds to promoters of phytochrome-regulated genes, thereby inhibiting FUM1 expression. PMID: 26949024
Q&A
Basic Research Questions
How to validate FUM1 antibody specificity in yeast mitochondrial studies?
Perform parallel Western blots using:
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Wild-type yeast lysates
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Mitochondrial vs cytosolic fractions (validate subcellular localization)
Example validation data from :
| Sample Type | Expected Band (kDa) | Observed Band (kDa) |
|---|---|---|
| Wild-type | 49.9 | 55 |
| fum2-1 mutant | No band | Absent |
Always include loading controls (e.g., anti-Pgk1 antibody) and test under both normal growth conditions and stress (400 mM hydroxyurea treatment) to confirm functional detection .
What methodological considerations are critical for immunofluorescence detection of FUM1?
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Fixation: Use paraformaldehyde ≤4% to preserve mitochondrial membrane integrity
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Permeabilization: Optimize digitonin vs Triton X-100 concentrations (0.1-0.3%)
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Mitotracker Red co-staining required for mitochondrial colocalization studies
Advanced Research Challenges
How to resolve contradictions in FUM1 post-translational modification studies?
Key conflict: Phosphorylation at T122/S124/T126 affects DNA repair function but not TCA cycle activity .
| Modification Site | Functional Impact | Conservation (Yeast vs Human) |
|---|---|---|
| T122/S124/T126 | DNA repair only | Fully conserved |
| K78/K79 | Both functions | Partially conserved |
Solution: Use phosphomimetic mutants (S→D) combined with succinylation-deficient strains ( , Fig 4a). Validate via tandem mass spectrometry and functional complementation assays on glycerol/HU plates.
How to address cross-reactivity between mitochondrial FUM1 and cytosolic isoforms?
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Design experiments using:
Critical finding: Anti-FUM1 antibodies from Agrisera (AS16 3966) detect both isoforms due to conserved epitopes – combine with genetic controls for unambiguous interpretation .
What orthogonal methods validate FUM1 antibody performance in structural studies?
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Cryo-EM validation pipeline:
Case study: Recombinant 139H2 antibody validation showed 92% structural congruence between computational models and cryo-EM density maps .
Methodological Innovation
How to integrate FUM1 antibody data with multi-omics datasets?
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Create unified analysis framework:
| Data Type | Tool | Key Parameter |
|---|---|---|
| Phosphoproteomics | MaxQuant | PTM localization probability |
| Metabolomics | MetaboAnalyst 5.0 | TCA cycle intermediate levels |
| Structural | ChimeraX | Epitope-antibody RMSD ≤2Å |
Cross-validate using FUM1 temperature-sensitive mutants and 13C-glucose flux analysis .
What emerging technologies complement traditional FUM1 antibody applications?
Data Interpretation Guidelines
How to troubleshoot non-reproducible Western blot bands?
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Systematic approach:
