GPAT4 Antibody
Description
Applications of GPAT4 Antibodies
GPAT4 antibodies are primarily employed in Western blotting (WB) to detect protein expression levels. Additional applications include:
Key Limitations:
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Limited reactivity to non-human/mouse species.
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Requires optimization for non-WB applications (e.g., immunoprecipitation).
Research Findings on GPAT4 Function
GPAT4 antibodies have elucidated critical roles in lipid metabolism, insulin signaling, and cellular homeostasis:
Role in Lipid Metabolism
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LPA Synthesis: GPAT4 converts glycerol-3-phosphate to LPA, a precursor for phospholipids and triacylglycerols (TAGs) .
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ER-Mitochondrial Crosstalk: Saturated LPA produced by GPAT4 inhibits autophagy via omegasome formation at ER-mitochondrial contact sites .
Insulin Resistance and Glucose Homeostasis
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Hepatic Insulin Resistance: Overexpression in hepatocytes reduces insulin-stimulated glycogen synthesis and Akt phosphorylation, impairing glucose metabolism .
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mTORC2 Regulation: GPAT4 suppresses mTORC2 activity by disrupting rictor association, linking lipid synthesis to insulin signaling .
Tissue-Specific Roles
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Testicular Development: Overexpression in GC-1spg cells (a spermatogenic cell line) increases proliferation via LPA-mediated GPCR signaling .
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Brown Adipose Tissue (BAT): Preferentially expressed in BAT, influencing thermogenesis and lipid storage .
Western Blotting Protocol
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Sample Preparation: Lyse cells in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.1% SDS) .
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SDS-PAGE: Resolve proteins on 8–10% gels.
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Blotting: Transfer to PVDF membranes.
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Primary Antibody: Incubate with GPAT4 antibody (1:1000–1:2000) at 4°C overnight.
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Detection: Use HRP-conjugated secondary antibodies and ECL substrates .
Troubleshooting:
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
Frequently Asked Questions (FAQs) for GPAT4 Antibody in Academic Research
Advanced Research Questions
How to resolve discrepancies in GPAT4 localization reports between ER and LDs?
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Stepwise strategy:
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Dynamic localization analysis: Treat cells with oleate to induce LD biogenesis. Monitor GPAT4 redistribution via live-cell imaging (e.g., GFP-tagged GPAT4) .
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Subcellular fractionation: Isolate ER and LD fractions via density-gradient centrifugation. Validate purity using organelle markers (e.g., calnexin for ER, PLIN2 for LDs).
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Knockdown validation: Deplete membrane-trafficking proteins (e.g., TRAPP complex components, COPII coat proteins) and quantify GPAT4 mislocalization using IF .
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How to design functional assays linking GPAT4 antibody-based detection to lipid metabolism?
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Integrated workflow:
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Lipidomic profiling: Combine GPAT4 immunoprecipitation with LC-MS to identify associated lipids (e.g., triacylglycerols, phosphatidic acid).
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CRISPR/Cas9 KO models: Compare lipid droplet size/distribution in GPAT4-KO vs. wild-type cells using antibody-stained confocal z-stacks .
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Rescue experiments: Overexpress GPAT4 mutants (e.g., hydrophobic hairpin deletion) and quantify LD morphology .
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Data Interpretation & Troubleshooting
How to address inconsistent GPAT4 antibody signals in cancer vs. metabolic disease models?
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Recommendations:
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Optimize antibody dilution ratios separately for each model.
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Include phosphatase/palmitoylation inhibitors during lysis to preserve modification-dependent epitopes.
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How to validate GPAT4’s role in ER-to-LD targeting using antibody-based assays?
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Key experiments:
