AGO7 Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Arabidopsis ARGONAUTE7 selects microRNA390 through multiple checkpoints during RISC assembly. PMID: 23732541
- AGO7's cytoplasmic and membranous siRNA body localization is essential for TAS3 precursor processing, highlighting a novel role for membrane-associated ribonucleoparticles in ta-siRNA biogenesis and AGO function in plants. PMID: 22327216
- miR390-ARGONAUTE7 complexes operate through distinct cleavage and non-cleavage mechanisms at two target sites within TAS3a transcripts. PMID: 18342362
Q&A
Here’s a structured FAQ for researchers working with AGO7 antibodies, synthesized from peer-reviewed studies and technical protocols:
What are the primary applications of AGO7 antibodies in plant molecular biology?
AGO7 antibodies are primarily used to:
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Localize AGO7 in cytoplasmic siRNA bodies via immunofluorescence (e.g., root meristem cells in Arabidopsis ).
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Validate protein interactions (e.g., colocalization with SGS3, RDR6, and miR390 in membrane-associated ribonucleoprotein complexes ).
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Assess functional relevance by comparing wild-type and mutant lines (e.g., ago7/zip-1 mutants ).
Methodological tip: Use anti-HA or anti-GFP tags under native promoters (pAGO7:HA-AGO7) to avoid overexpression artifacts .
How do I validate AGO7 antibody specificity in immunoassays?
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Negative controls: Test in ago7 mutant backgrounds to confirm signal loss .
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Cross-reactivity checks: Compare with other AGO family members (AGO1, AGO4) using epitope-tagged proteins .
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Functional rescue: Confirm antibody specificity by reintroducing tagged AGO7 into mutants and verifying phenotypic reversion .
Example validation data:
| Assay | Wild-Type Signal | ago7 Mutant Signal |
|---|---|---|
| IF (Anti-HA) | Strong cytoplasmic foci | No signal |
| WB (Anti-GFP) | ~130 kDa band | No band |
How to resolve contradictions in AGO7 subcellular localization studies?
Conflicting reports of nuclear vs. cytoplasmic AGO7 localization often arise from:
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Tag placement: N-terminal tags (GFP/HA) preserve function, while C-terminal tags may disrupt siRNA body formation .
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Stress conditions: Stress granules merge with siRNA bodies under translational repression, altering localization .
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Artifacts from overexpression: Use native promoters (pAGO7) instead of constitutive promoters (e.g., p35S) .
Solution: Combine immunolocalization with subcellular fractionation (e.g., microsomal membrane isolation) to confirm membrane association .
What methods optimize co-immunoprecipitation of AGO7 complexes?
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Lysis buffer: Include 1% Triton X-100 + RNase inhibitors to preserve RNA-protein interactions .
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Chaperone dependence: Pre-treat with Hsp70/Hsp90 inhibitors (e.g., geldanamycin) to assess RISC assembly requirements .
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Crosslinking: Use formaldehyde (1%) for transient interactions (e.g., AGO7-miR390 binding ).
Critical step: Validate co-IPs with in vitro reconstitution assays using plant cell-free lysates .
Why does AGO7 antibody staining show nucleolar signals in some studies?
AGO7 exhibits stress-dependent nuclear shuttling:
Interpretation framework:
| Condition | Localization Pattern | Frequency |
|---|---|---|
| Wild-Type | Cytoplasmic foci | 95% |
| NLS-AGO7 | Nuclear | 100% |
How to address low signal-to-noise ratios in AGO7 Western blots?
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Antibody dilution: Use anti-AGO7 at 1:1,000 + secondary HRP-conjugated antibody (1:25,000) .
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Blocking: 5% BSA + 0.1% Tween-20 reduces non-specific binding in plant extracts .
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Positive control: Include pAGO7:HA-AGO7 transgenic lines to confirm antibody reactivity .
Can AGO7 antibodies be repurposed for studying viral pathogenesis?
Yes, for:
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Viral protein interactions: AGO7 colocalizes with TEV VP6 in membrane-associated complexes .
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Host defense mechanisms: Monitor AGO7 redistribution during infection (e.g., stress granule markers like TIA-1 ).
Experimental design: Compare wild-type and ago7 mutants for viral susceptibility .
