AMI1 Antibody
Description
Introduction to AMI1 Antibody
The AMI1 antibody targets the amidase_3 domain-containing protein Ami1, a critical enzyme involved in peptidoglycan (PG) hydrolysis and cell wall metabolism in mycobacteria. AMI1 plays a role in bacterial growth, cell division, and virulence, particularly in Mycobacterium abscessus (M. abscessus) and Mycobacterium tuberculosis (M. tuberculosis). Research highlights its dispensability for in vitro growth but underscores its importance in cell wall permeability, antibiotic susceptibility, and pathogenicity .
Structure and Functional Role of Ami1
Ami1 is a zinc-dependent PG hydrolase characterized by its amidase_3 domain. Key structural and functional insights include:
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Domain Architecture: Comprises an amidase_3 catalytic domain responsible for cleaving PG bonds .
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Biological Role:
In Vitro and In Vivo Studies
| Parameter | Wild-Type M. abscessus | Δami1 Mutant | Complemented Strain |
|---|---|---|---|
| Growth in Broth Medium | Normal | No defect | Restored to wild-type |
| Cell Division | Regular septation | Impaired | Partially restored |
| Virulence (Zebrafish) | High pathogenicity | Attenuated | Partially restored |
| Biofilm Formation | Robust | Reduced | Not reported |
Data derived from M. abscessus S and R morphotypes showed that ami1 deletion does not impair in vitro growth but reduces virulence and disrupts cell division .
Biochemical Characterization
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Enzymatic Activity: Purified Ami1 hydrolyzes PG fragments in a zinc-dependent manner .
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Genetic Complementation: Expression of ami1 from M. tuberculosis (Rv3717) rescues cell division defects in M. smegmatis Δami1 mutants .
Comparative Analysis of Ami1 Across Species
| Species | Role of Ami1 | Phenotype of Δami1 Mutant |
|---|---|---|
| M. abscessus | Cell separation, virulence | Attenuated infection in macrophages |
| M. smegmatis | Septal PG turnover, biofilm formation | Increased antibiotic susceptibility |
| M. tuberculosis | Chronic infection persistence | Not fully characterized |
Implications for Therapeutic Development
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Antibiotic Adjuvant Potential: AMI1 deletion increases cell wall permeability, enhancing susceptibility to β-lactams and vancomycin .
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Vaccine Target: While not a direct vaccine candidate, understanding AMI1’s role in virulence could inform anti-mycobacterial strategies .
Challenges and Future Directions
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- An analysis of amidase 1 in rat and Arabidopsis (PMID: 17555521)
Q&A
Basic Research Questions
How to determine AMI1 antibody specificity for amyloid protofibrils versus other conformational states?
Validate specificity using a combination of:
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ELISA: Compare binding to protofibrils, monomers, and fibrils (e.g., Aβ42 protofibrils vs. Aβ40 monomers) .
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Immunocytochemistry: Test reactivity in HEK293 cells expressing wild-type vs. mutated amyloid constructs .
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Aggregation inhibition assays: Monitor changes in Thioflavin T fluorescence kinetics when AMI1 is introduced during amyloid formation .
What experimental models are suitable for assessing AMI1’s in vivo efficacy?
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Transgenic mouse models: Use animals expressing human amyloid proteins (e.g., APP/PS1 for Alzheimer’s disease) to evaluate antibody penetration and plaque reduction .
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Primary neuronal cultures: Measure miniature excitatory postsynaptic currents (mEPSCs) to assess functional effects on synaptic transmission .
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Immunohistochemistry: Compare antibody labeling patterns in diseased vs. healthy brain tissue sections .
How to optimize AMI1 purification for research applications?
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Alternative resins: Replace Protein A with cation-exchange chromatography or precipitation-based methods to reduce costs while maintaining purity .
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Quality control: Use SDS-PAGE and size-exclusion chromatography to verify monomeric antibody integrity.
Advanced Research Questions
How to resolve discrepancies between in vitro binding data and in vivo efficacy?
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Troubleshooting steps:
What strategies can map AMI1’s conformational epitope on amyloid protofibrils?
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Phage display libraries: Identify peptide sequences that mimic the epitope.
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Cryo-EM or X-ray crystallography: Resolve the antibody-antigen complex structure at high resolution .
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Alanine scanning mutagenesis: Systematically mutate residues in the amyloid sequence to pinpoint critical binding regions.
How to design experiments assessing AMI1’s cross-reactivity with non-target amyloids?
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Comparative binding assays: Test reactivity against α-synuclein, tau, or transthyretin protofibrils using surface plasmon resonance (SPR) .
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Functional inhibition assays: Measure AMI1’s ability to suppress aggregation of diverse amyloids via turbidity or fluorescence assays.
Methodological Insights from Published Data
Table 1: AMI1 Reactivity Across Amyloid Types
What controls are critical for ensuring reproducibility in AMI1-based assays?
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Include isotype-matched negative controls in all binding experiments.
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Standardize protofibril preparation using techniques like dynamic light scattering (DLS) to confirm particle size .
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Use AMPAR knockout neuronal cultures to isolate AMI1-specific effects in functional studies .
How to address lot-to-lot variability in AMI1 production for longitudinal studies?
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Implement a stability-indicating assay (e.g., differential scanning fluorimetry) to monitor aggregation-prone regions.
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Maintain a centralized repository for antibody aliquots to minimize storage-related degradation .
Addressing Data Contradictions
How to interpret conflicting results between Western blot and immunocytochemistry data?
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Potential causes:
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Epitope masking in denatured samples (Western blot) vs. native conformation (ICC).
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Cross-reactivity with unrelated proteins in lysate preparations.
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Resolution: Perform FP-based Western blots with fusion proteins retaining native epitopes .
