At3g05500 Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Q&A
Basic Research Questions
What experimental applications are validated for At3g05500 antibodies in Arabidopsis studies?
The At3g05500 antibody (UniProt Q9MA63) is validated for:
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Western blot (WB): Detects the ~27 kDa REF/SRPP-like protein under denaturing conditions .
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ELISA: Quantifies recombinant At3g05500 protein with high specificity .
Methodological note: Always include recombinant At3g05500 protein (e.g., MBS1430164 ) as a positive control and Arabidopsis mutant lines lacking At3g05500 as negative controls.
How should researchers validate antibody specificity for At3g05500?
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Perform immunoblotting with Arabidopsis wild-type vs. at3g05500 knockout lysates .
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Use antigen pre-absorption assays: Preincubate antibody with recombinant At3g05500 protein (1:5 molar ratio) to confirm signal loss .
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Cross-reference with mass spectrometry data from immunoprecipitated samples.
How to address non-specific binding in WB when studying lipid droplet-associated proteins?
What methodologies resolve contradictions in At3g05500 expression data across studies?
Common discrepancies arise from:
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Growth conditions: Light/dark cycles alter lipid droplet dynamics in Arabidopsis . Standardize to 16h light/8h dark.
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Antibody batch variation: Compare lot-specific QC reports (e.g., ≥85% purity by SDS-PAGE ).
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Protein extraction: Use 8M urea + 2% CHAPS buffer to improve solubility of hydrophobic REF-family proteins .
What orthogonal methods confirm At3g05500 subcellular localization?
Combine antibody-based detection with:
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Confocal microscopy: Express At3g05500-GFP under native promoter
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Cellular fractionation: Validate lipid droplet enrichment via sucrose density gradient centrifugation .
How to optimize co-immunoprecipitation (Co-IP) for At3g05500 interactome studies?
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Use crosslinkers: 1 mM DSP for 30 min at 4°C to stabilize transient interactions
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Elution buffer: 100 mM glycine (pH 2.5) + 0.1% Triton X-114
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Proteomic controls: Include empty bead samples to exclude nonspecific binders.
What statistical approaches are recommended for qPCR/WB correlation studies?
Apply mixed-effects models to account for:
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Biological vs technical replicates
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Batch effects across antibody lots
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Non-normal distributions (use Box-Cox transformations)
