GH3.3 Antibody
Description
GHProtein Overview
GH3.3 belongs to the GH3 family of acyl acid-amido synthetases, primarily functioning in auxin homeostasis by conjugating indole-3-acetic acid (IAA) with amino acids like aspartate (Asp) or glutamate (Glu) . Key characteristics include:
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Substrate specificity: Preferential activity toward IAA over synthetic auxins like 2,4-D .
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Enzymatic efficiency: Kinetic parameters vary across GH3 isoforms (Table 1) .
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Regulation: Expression is modulated by transcription factors TCP20 and WRI1 in Arabidopsis roots .
Table 1: Kinetic Parameters of Select GH3 Proteins
Anti-GHDC Antibody (A12411)
This polyclonal IgG antibody targets the GH3 domain-containing protein GHDC, which shares structural motifs with GH3 family members .
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Applications: Validated for Western blot (0.5–1 µg/mL), immunofluorescence (20 µg/mL), and ELISA .
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Cross-reactivity: Reacts with human, mouse, and rat homologs .
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Molecular weight: Detects ~68 kDa protein in human cell lysates .
Monoclonal Antibodies in GH3 Cell Research
The rat pituitary cell line GH3 has been used to study antibodies against epidermal growth factor receptor (EGFR). For example:
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2G2-IgM: Induces prolactin synthesis in GH3 cells by mimicking EGFR activation .
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Specificity: Binds EGFR’s extracellular domain, triggering downstream signaling akin to EGF .
Promoter Analysis
The GH3.3 promoter’s auxin-responsive element (AuxRE) has been dissected using GUS reporter fusions. A 300 bp proximal region is essential for auxin-induced expression .
Functional Studies
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Loss-of-function mutants: tcp20 mutants show downregulated GH3.3 expression, implicating TCP20 in transcriptional activation .
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Overexpression: Increased GH3.3 activity elevates IAA-Asp conjugates, altering auxin signaling dynamics .
Challenges and Opportunities
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Antibody specificity: No commercially available antibodies exclusively targeting GH33 are documented; most studies rely on genetic constructs (e.g., promoter-GUS fusions) .
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Therapeutic potential: Broadly reactive IgG3 antibodies (e.g., targeting viral glycans) highlight the utility of GH3 domain-related immunogens in vaccine design .
Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- The GH3.3 gene plays a crucial role in fine-tuning adventitious root initiation in the Arabidopsis thaliana hypocotyl, and its function is mediated by modulation of jasmonic acid homeostasis. PMID: 22730403
Q&A
FAQs for GH3.3 Antibody in Academic Research
How is antibody specificity validated for GH3.3 in plant hormone studies?
To validate GH3.3 antibody specificity:
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Western Blot: Use lysates from transgenic lines overexpressing GH3.3 and knockout mutants. A single band at the predicted molecular weight (e.g., ~65 kDa for Arabidopsis GH3 proteins) confirms specificity .
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Pre-absorption Control: Pre-incubate the antibody with purified GH3.3 protein; signal loss confirms target specificity.
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Cross-Reactivity Testing: Test against lysates expressing homologous GH3 family members (e.g., GH3.5, GH3.12) to rule out off-target binding .
Example Validation Data:
| Technique | Sample | Expected Band (kDa) | Observed Band (kDa) |
|---|---|---|---|
| Western Blot | Arabidopsis GH3.3-OE | 65 | 65 |
| Western Blot | gh3.3 mutant | 65 | No band |
What experimental designs are optimal for studying GH3.3’s role in jasmonate signaling?
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Time-Course Treatments: Apply jasmonic acid (JA) or its precursors (e.g., OPDA) to wild-type and gh3.3 mutants, then quantify GH3.3 protein levels via immunoassays .
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Co-Immunoprecipitation (Co-IP): Identify GH3.3 interaction partners (e.g., JAZ repressors) using anti-GH3.3 antibodies coupled with mass spectrometry .
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Localization Studies: Perform immunofluorescence or GFP-tagged GH3.3 imaging to track subcellular dynamics under stress conditions.
Critical Controls:
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Include gh3.3 knockout lines to confirm antibody-dependent signals.
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Use JA biosynthesis inhibitors (e.g., ibuprofen) to validate hormone-specific responses .
How to resolve discrepancies in GH3.3 molecular weight across studies?
Observed MW variations (e.g., 65 vs. 70 kDa) may arise from:
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Post-Translational Modifications: Test for phosphorylation using λ-phosphatase treatment .
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Alternative Splicing: Perform RNA-seq on experimental samples to detect splice variants.
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Gel Conditions: Compare reducing vs. non-reducing SDS-PAGE (e.g., β-mercaptoethanol alters disulfide bonds) .
Case Study:
In Galectin-3 studies, reducing conditions shifted observed MW from 28 kDa (monomer) to 37–38 kDa (dimer) . Apply similar troubleshooting for GH3.3.
What functional assays confirm GH3.3 antibody activity beyond binding?
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Enzyme Activity Inhibition: Pre-incubate GH3.3 protein with antibodies and measure JA-Ile synthetase activity via LC-MS .
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Phenotypic Rescue: Microinject GH3.3 antibodies into gh3.3 mutants and assess JA-responsive gene expression (e.g., VSP2, LOX2) .
Data Interpretation:
| Assay | Expected Outcome (Antibody+) | Negative Control (Antibody–) |
|---|---|---|
| JA-Ile Synthesis | Reduced conjugate formation | Normal activity |
| VSP2 Expression | Suppressed induction | Wild-type levels |
How to optimize GH3.3 detection in complex plant tissues?
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Sample Preparation: Use fresh-frozen tissues to avoid epitope degradation. For woody tissues, add polyvinylpyrrolidone to reduce phenolic interference.
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Signal Amplification: Employ tyramide-based amplification in immunohistochemistry for low-abundance targets .
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Multiplexing: Pair GH3.3 antibodies with hormone biosensors (e.g., DR5:GFP for auxin) in dual-labeling experiments.
Troubleshooting Table:
| Issue | Solution |
|---|---|
| High background | Block with 5% non-fat milk + 0.1% Tween-20 |
| Weak signal | Increase primary antibody incubation time (e.g., 48 hr at 4°C) |
