GATL5 Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research indicates that GALACTURONOSYLTRANSFERASE-LIKE5 (AtGATL5) may play a regulatory role in determining the final size of the mucilage rhamnogalacturonan I. PMID: 24092888
Q&A
Here’s a structured FAQ collection for GATL5 antibody research, synthesized from peer-reviewed methodologies and principles in immunology, antibody production, and experimental design:
How should researchers design experiments to evaluate GATL5 antibody specificity in cellular assays?
To ensure specificity:
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Pre-staining controls: Include unstained cells, isotype controls, and fluorescence-minus-one (FMO) controls to distinguish background noise .
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Fixation compatibility: Test antibody binding pre- and post-fixation, as some fixatives (e.g., paraformaldehyde) may alter epitope accessibility .
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Blocking steps: Use Fc receptor blockers (e.g., anti-CD16/32) to minimize non-specific binding in immune cell assays .
Example workflow:
What factors influence GATL5 antibody performance in Western blotting?
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Antigen preparation: Use purified protein lysates to avoid cross-reactivity with impurities (e.g., endotoxins) .
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Antibody dilution: Optimize using a titration series (e.g., 1:500–1:5,000) in 5% BSA/TBST .
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Validation: Confirm target band size with knockout cell lines or siRNA knockdown .
How to standardize GATL5 antibody dosing in animal models?
Refer to species-specific guidelines:
| Animal | Adjuvant Type | Max Volume (Oil-Based) | Route |
|---|---|---|---|
| Mice | Freund’s | 100 μL | s.c. |
| Rabbits | Freund’s | 1 mL | i.d. |
| Goats | Alum | 5 mL | i.m. |
| Data adapted from immunization protocols |
How to resolve contradictory GATL5 antibody binding data across studies?
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Antigen purity: Trace impurities (<1%) in immunogen preparation can dominate immune responses, leading to off-target antibodies . Validate antigen purity via SDS-PAGE and mass spectrometry.
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Host variability: Anti-glycan antibodies (e.g., anti-Neu5Gc) show gender-based differences (higher IgG in males) . Control for host factors (age, sex, geographic origin) in cohort studies .
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Epitope mapping: Use peptide arrays or hydrogen-deuterium exchange mass spectrometry (HDX-MS) to confirm binding regions .
What strategies mitigate cross-reactivity in GATL5 antibody-based assays?
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Pre-absorption: Incubate antibodies with non-target tissues/cell lysates to remove cross-reactive clones .
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Glycan masking: Treat samples with neuraminidase to expose/hide glycan epitopes (e.g., α-Gal) .
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Multiplex validation: Combine ELISA, flow cytometry, and immunohistochemistry to confirm consistency .
How to optimize GATL5 antibody production for high-affinity monoclonal clones?
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Adjuvant selection: Compare antibody titers using Freund’s vs. alum adjuvants (Freund’s enhances Th1 responses) .
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B-cell sequencing: Use Ig-seq to identify high-affinity clones from peripheral blood or lymph nodes .
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Hybridoma screening: Prioritize clones with >80% binding inhibition in competitive ELISA .
Methodological Considerations for Data Reproducibility
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Antibody lot variability: Batch-test new lots using standardized positive/negative controls .
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Long-term storage: Aliquot antibodies in stabilizing buffers (e.g., 50% glycerol + 0.1% sodium azide) at -80°C .
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Quantitative thresholds: Define positivity thresholds using ROC curve analysis (e.g., ≥2x background MFI in flow cytometry) .
