GH3.5 Antibody
Description
Target Protein Overview
The GH3.5 antibody (Catalog: A12411) specifically detects GH3 domain-containing protein (GHDC), a 57.5 kDa protein encoded by the GHDC gene in humans . Alternative names include LGP1 and D11LGP1, with roles hypothesized in enzymatic or regulatory functions due to its GH3 domain architecture .
Key Parameters:
Western Blot Performance:
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Cell Lysate: Detected GHDC in 293 cells at both 0.5 μg/mL and 1 μg/mL, showing concentration-dependent band intensity .
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Specificity: No cross-reactivity reported under tested conditions .
Immunofluorescence:
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Localized GHDC in human breast tissue sections at 20 μg/mL, demonstrating clear cytoplasmic staining .
Functional Context of GH3 Proteins
While the GH3.5 antibody targets human GHDC, the broader GH3 family includes proteins like Arabidopsis thaliana GH3.5, which regulates plant hormone signaling:
Technical Considerations for Antibody Use
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Optimization: Dilutions are starting points; end-user titration is critical due to variability in sample preparation and detection systems .
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Limitations: No data for non-human species or applications beyond WB/IF/ELISA .
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Cross-Reactivity Note: Despite naming similarities, this antibody does not target plant GH3.5 proteins .
Research Implications
The GH3.5 antibody enables studies on GHDC’s role in human physiology, though its exact mechanistic functions remain underexplored. In contrast, plant GH3.5 is well-characterized as a bifunctional enzyme modulating salicylic acid (SA)-dependent defense and auxin-mediated growth . For example:
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Kinetic Preference: Arabidopsis GH3.5 conjugates indole-3-acetic acid (IAA) to aspartate 360× more efficiently than SA under high aspartate conditions, favoring growth regulation .
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Pathogen Response: Overexpression of GH3.5 in plants elevates SA but paradoxically increases susceptibility by dysregulating auxin signaling .
Outstanding Questions
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What molecular interactions does human GHDC mediate?
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Does GHDC exhibit enzymatic activity akin to plant GH3.5?
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Are there clinical correlations between GHDC expression and human diseases?
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research suggests that AtGH3.5 conjugates auxins (e.g., IAA and PAA) and benzoates (e.g., SA and BA) to regulate crosstalk between different metabolic pathways, expanding the potential roles of GH3 acyl acid amido synthetases in plants. PMID: 27849615
- Arabidopsis acetyl-amido synthetase GH3.5 plays a role in camalexin biosynthesis by conjugating indole-3-carboxylic acid and cysteine, and upregulating camalexin biosynthesis genes. PMID: 22624950
- The GH3.5 gene is essential for fine-tuning adventitious root initiation in the Arabidopsis thaliana hypocotyl, acting by modulating jasmonic acid homeostasis. PMID: 22730403
- Findings support the role of WES1 in regulating hypocotyl growth by mediating phytochrome B-perceived light signals. PMID: 17602188
- This study demonstrates that GH3.5 acts as a bifunctional modulator in both salicylic acid and auxin signaling during pathogen infection. PMID: 17704230
Q&A
How can researchers validate GH3.5 antibody specificity in Arabidopsis thaliana?
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Methodological approach:
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Use knockout mutants (e.g., wes1 or gh3.5-1D lines) as negative controls in Western blot (WB) or immunofluorescence (IF) to confirm absence of signal .
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Perform peptide competition assays by pre-incubating the antibody with its immunogen peptide (e.g., 19-aa peptide for GHDC antibody) .
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Validate cross-reactivity using tissues from orthologous species (e.g., mouse or rat) if working with non-Arabidopsis systems .
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Compare results with independent detection methods (e.g., mRNA quantification via qRT-PCR or enzymatic activity assays) .
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What experimental controls are critical for GH3.5 antibody applications in hormone signaling studies?
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Essential controls:
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Biological replicates across hormone-treated vs. untreated plants (e.g., IAA, SA, or pathogen-infected samples) .
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Technical controls:
Control Type Purpose Example No-primary-antibody Rule out nonspecific binding Omit GH3.5 antibody in IF Isotype control Confirm antibody specificity Use rabbit IgG in WB -
Include internal standards (e.g., housekeeping proteins like actin) for normalization in WB .
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How does GH3.5 antibody facilitate analysis of auxin-SA crosstalk in plant-pathogen interactions?
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Advanced methodology:
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Quantify GH3.5 protein levels in pathogen-challenged tissues (e.g., Pseudomonas syringae-infected leaves) using IF or WB .
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Correlate GH3.5 expression with hormone metabolites (e.g., free IAA, SA, and their conjugates) via LC-MS .
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Combine with transgenic lines (e.g., gh3.5-1D overexpression) to assess altered hormone gradients and PR-1 gene expression .
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How to resolve contradictory data in GH3.5 subcellular localization studies?
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Troubleshooting strategies:
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Optimize fixation and permeabilization protocols for IF (e.g., test 4% paraformaldehyde vs. methanol) .
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Validate with subcellular fractionation followed by WB to confirm nuclear vs. cytoplasmic localization .
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Account for tissue-specific expression: GH3.5 may localize differently in roots vs. leaves under stress .
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What are the functional implications of GH3.5’s dual substrate specificity?
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Biochemical insights:
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Experimental design: Use GH3.5 antibody to monitor enzyme abundance in mutants lacking other GH3 family members (e.g., GH3.1, GH3.6) to isolate its unique roles .
How to optimize GH3.5 antibody dilution for low-abundance targets?
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Protocol refinement:
What molecular tools complement GH3.5 antibody in studying its bifunctionality?
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Integrated approaches:
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Crystallography: Resolve GH3.5 structure (e.g., IAA/AMP-bound conformations) to identify substrate-binding residues .
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Kinetic assays: Measure enzyme activity toward IAA vs. SA using recombinant GH3.5 and radiolabeled ATP .
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Transcriptomics: Pair antibody-based protein data with RNA-seq of GH3.5-overexpressing lines to identify downstream targets .
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