AT6 Antibody
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
What experimental applications is the AT6/172 antibody validated for, and what evidence supports this?
The AT6/172 antibody is validated for immunocytochemistry (ICC) and western blotting (WB) under standardized protocols . Validation evidence includes:
For ICC, researchers should use 1–5 µg/mL in PBS/1% BSA with methanol fixation . For WB, 0.5–2 µg/mL in TBST/5% non-fat milk is recommended, with verification against recombinant α-actinin .
How should researchers validate species cross-reactivity for novel model systems?
Cross-reactivity beyond the documented species (human, mouse, rabbit) requires:
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Phylogenetic alignment: Compare α-actinin epitope regions (AA 150–300) across species using tools like Clustal Omega .
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Empirical testing: Perform WB on lysates from the novel species alongside positive controls.
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Competition assays: Pre-incubate antibody with purified human α-actinin to confirm signal loss .
A study on rabbit cardiac tissue demonstrated 89% sequence homology at the AT6/172 epitope, enabling cross-reactivity confirmation via peptide blocking .
What controls are essential when using AT6/172 in subcellular localization studies?
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Knockout validation: CRISPR-edited α-actinin-null cells (e.g., HEK293 ΔACTN4) .
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Compartmental markers: Co-staining with β-tubulin (cytoplasmic) or lamin B1 (nuclear) .
In cardiac myocytes, AT6/172 localized α-actinin to Z-discs only when fixation included 4% PFA + 0.1% glutaraldehyde, emphasizing protocol standardization .
How can researchers resolve contradictory data between WB and ICC results using AT6/172?
Contradictions often arise from post-translational modifications (PTMs) or epitope masking:
| Scenario | WB Result | ICC Result | Resolution Strategy |
|---|---|---|---|
| Strong WB, weak ICC | Clear ~100 kDa | Faint signal | Optimize permeabilization (e.g., 0.2% Triton) |
| Weak WB, strong ICC | Faint band | Robust staining | Check phosphorylation (e.g., Ser/Thr sites) |
A 2021 study resolved discordance in vascular smooth muscle cells by treating lysates with λ-phosphatase, restoring WB-ICC correlation .
What strategies enable multiplexed assays combining AT6/172 with other markers?
Sequential staining protocol
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Fixation: 4% PFA, 10 min
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Primary antibodies: AT6/172 (mouse IgG1) + β-catenin (rabbit IgG)
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Secondary reagents:
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Anti-mouse AF488 (1:500)
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Anti-rabbit AF594 (1:1,000)
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Mounting: ProLong Diamond with DAPI
Validation requirements
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Spectral unmixing validation for super-resolution microscopy
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Cross-adsorption checks for secondary antibodies
A 2023 multiplex study achieved <40 nm resolution in podocytes using STED microscopy with AT6/172 .
How does the antibody’s performance vary in disease models like cardiac hypertrophy?
In pressure-overloaded mouse hearts:
| Parameter | Hypertrophy Model (TAC) | Sham Control | p-value |
|---|---|---|---|
| AT6/172 signal intensity | 2.3 ± 0.4 AU | 1.0 ± 0.2 AU | <0.001 |
| Sarcomere localization | Disrupted (43% cells) | Organized (92%) | <0.01 |
Key methodological considerations:
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Decalcification time ≤24 hr to preserve epitopes
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Antigen retrieval: 10 mM citrate buffer, pH 6.0, 95°C, 20 min
What computational tools enhance data interpretation from AT6/172-based experiments?
Image analysis pipeline
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Preprocessing: Fiji/ImageJ background subtraction (rolling ball radius=50 px)
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Segmentation: Trainable Weka Segmentation for Z-disc identification
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Quantification:
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Fiber orientation entropy (FiberFit plugin)
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Co-localization with integrins (Manders’ coefficient)
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Statistical framework
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Mixed-effects models for longitudinal studies
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Bayesian hierarchical modeling for multi-experiment data
A 2024 study integrating AT6/172 data with RNA-seq revealed ACTN2-ITGB1 co-regulation (ρ=0.72, FDR<0.05) in failing hearts .
