AIM25 Antibody

What is AIM2 and what cellular functions does it perform?

AIM2 is a 343 amino acid (39 kDa) protein with a N-terminal DAPIN (pyrin) domain (amino acids 1-87) and a C-terminal HIN-200 domain (amino acids 138-337), which contains two oligonucleotide-binding folds. AIM2 functions as a critical component of the inflammasome, participating in innate immune response by recognizing cytosolic double-stranded DNA. Upon binding to DNA, AIM2 undergoes oligomerization and associates with PYCARD, initiating the recruitment of caspase-1 precursors and processing of IL-1β and IL-18 . This mechanism represents a fundamental pathway in the cellular response to pathogens and cellular damage, making AIM2 a significant target for immunological research.

What are the optimal storage conditions for maintaining AIM2 antibody activity?

For maximum stability and performance, AIM2 antibodies should be stored according to the following protocol:

• Store at -20 to -70°C for up to 12 months from date of receipt, in the supplied condition

• After opening, store at 2 to 8°C under sterile conditions for up to 1 month

• For longer storage after opening, maintain at -20 to -70°C under sterile conditions for up to 6 months

• Use a manual defrost freezer and strictly avoid repeated freeze-thaw cycles which significantly impact antibody performance

These guidelines ensure optimal antibody activity across experimental timeframes and maintain consistent performance between experiments.

What applications have AIM2 antibodies been validated for?

AIM2 antibodies have been validated for multiple experimental applications, with different clones offering specific utility profiles:

Selection of the appropriate antibody should be based on the intended application and target species requirements .

How can researchers optimize Western blot protocols for AIM2 detection?

Optimizing Western blot protocols for AIM2 detection requires careful consideration of several technical parameters:

• Sample preparation: Use PVDF membrane for optimal protein binding characteristics with AIM2

• Antibody concentration: Begin with 2 μg/mL of anti-AIM2 antibody for initial detection

• Secondary antibody selection: HRP-conjugated anti-rabbit IgG (for rabbit primary antibodies) has demonstrated good signal-to-noise ratio

• Expected molecular weight: AIM2 typically appears at approximately 40 kDa under reducing conditions

• Positive control recommendation: Include Daudi human Burkitt's lymphoma cell line lysate as a validated positive control

• Buffer system: Utilize Immunoblot Buffer Group 1 for optimal results under reducing conditions

Systematic optimization of these parameters is essential for generating reproducible and specific AIM2 detection across different experimental contexts.

What experimental approaches are most effective for studying AIM2 inflammasome activation?

Investigating AIM2 inflammasome activation requires multi-faceted methodological approaches:

• DNA-mediated activation models:

  • Transfect synthetic double-stranded DNA of various lengths

  • Apply pathogen-derived DNA (bacterial, viral) for physiologically relevant activation

  • Monitor kinetics of activation through time-course experiments

• Protein interaction analysis:

  • Co-immunoprecipitation of AIM2 with PYCARD/ASC adapter protein

  • Proximity ligation assays to visualize in situ protein-protein interactions

  • FRET-based approaches for real-time interaction monitoring

• Functional readouts:

  • Western blot detection of cleaved caspase-1 (p20 subunit)

  • ELISA measurement of secreted IL-1β and IL-18

  • Cell death assays to quantify pyroptosis

These approaches provide complementary data on both the molecular assembly and functional consequences of AIM2 activation .

How should researchers design experiments to investigate AIM2's role in inflammatory diseases like rheumatoid arthritis?

Investigating AIM2's role in rheumatoid arthritis requires a strategic experimental design framework:

• Patient sample analysis:

  • Quantify AIM2 expression in synovial tissues and fibroblast-like synoviocytes (FLS)

  • Compare expression profiles between RA patients and appropriate controls

  • Correlate expression with clinical parameters and disease severity

• Mechanistic investigations:

  • Examine AIM2 inflammasome assembly in primary FLS cultures

  • Assess DNA-sensing capacity in the synovial microenvironment

  • Determine downstream inflammatory mediator profiles

• Intervention approaches:

  • siRNA or CRISPR-based knockout studies in cellular models

  • Inhibitor studies targeting the AIM2 pathway

  • Monitor pathway activity in response to conventional RA therapeutics

Evidence suggests AIM2 plays a significant role in FLS-mediated inflammation in rheumatoid arthritis contexts, warranting systematic investigation using these methodological approaches .

What controls should be included when validating a new AIM2 antibody?

Comprehensive validation of a new AIM2 antibody requires inclusion of several critical controls:

• Positive expression controls:

  • Daudi human Burkitt's lymphoma cell line (validated AIM2 expressor)

  • Interferon-stimulated cells (AIM2 is interferon-inducible)

  • Recombinant AIM2 protein (for absolute specificity determination)

• Negative controls:

  • AIM2 knockdown/knockout cell lines (CRISPR or siRNA)

  • Cell types with confirmed absence of AIM2 expression

  • Secondary antibody-only controls to assess non-specific binding

• Specificity controls:

  • Peptide competition assays using the immunizing peptide

  • Multiple antibodies targeting different AIM2 epitopes

  • Cross-reactivity testing with related HIN-200 family proteins

Implementing this control framework ensures confidence in antibody specificity and performance across experimental applications .

How can researchers distinguish between specific and non-specific signals when using AIM2 antibodies?

Distinguishing specific from non-specific signals requires systematic analytical approaches:

• Signal verification methods:

  • Comparing multiple antibodies targeting different AIM2 regions (N-terminal DAPIN domain versus C-terminal HIN-200 domain)

  • Validating signal molecular weight (expected 40 kDa for full-length AIM2)

  • Confirming signal reduction/elimination in AIM2-depleted samples

• Optimization parameters for reducing non-specific binding:

  • Titration of primary antibody concentration (0.5-5 μg/mL range)

  • Extended washing steps with appropriate buffers

  • Alternative blocking agents (BSA, milk, commercial blockers)

  • Varying incubation temperatures and durations

• Technical considerations:

  • Use of highly purified antibody preparations

  • Selection of detection systems with optimal signal-to-noise characteristics

  • Implementation of digital image analysis with background subtraction

These strategies systematically address the common sources of non-specific signals in antibody-based detection systems .

What approaches allow differentiation between AIM2 and other HIN-200 family proteins?

Differentiating AIM2 from other structurally related HIN-200 family members requires specialized approaches:

• Epitope selection strategy:

  • Choose antibodies targeting regions with minimal sequence homology to other HIN-200 proteins

  • Human AIM2 shares 90% amino acid sequence identity with macaque AIM2 over the aa 71-180 region, but has lower homology with other family members

• Validation methodologies:

  • Mass spectrometry confirmation of immunoprecipitated proteins

  • Parallel detection with antibodies against multiple HIN-200 family members

  • Cross-reactivity testing against recombinant HIN-200 proteins

• Functional discrimination:

  • DNA-binding capacity assessment (characteristic of AIM2)

  • Subcellular localization studies (nuclear vs. cytoplasmic)

  • Pathway-specific activation profiles

These approaches enable confident discrimination between AIM2 and structurally related proteins, essential for accurate experimental interpretation.

How can researchers quantify AIM2 expression and activation across different experimental conditions?

Rigorous quantification of AIM2 expression and activation requires standardized analytical methods:

• Expression quantification:

  • Densitometric analysis of Western blots with appropriate normalization

  • qPCR measurement of AIM2 mRNA (normalized to reference genes)

  • Flow cytometric quantification of per-cell protein levels

• Activation assessment:

  • Oligomerization state analysis through native gel electrophoresis

  • Co-immunoprecipitation efficiency with PYCARD/ASC

  • Downstream functional readouts (caspase-1 cleavage, IL-1β/IL-18 secretion)

• Quantification parameters:

  • Standard curve generation using recombinant proteins

  • Multiple technical and biological replicates

  • Statistical analysis appropriate for data distribution

This multi-parameter approach provides comprehensive assessment of both AIM2 abundance and functional status across experimental conditions .

What are promising research directions for AIM2 antibodies in immunological disorders?

Emerging evidence suggests several high-potential research avenues:

• Role in autoimmune pathologies:

  • Beyond rheumatoid arthritis, AIM2's involvement in systemic lupus erythematosus, psoriasis, and inflammatory bowel disease warrants investigation

  • Potential as a biomarker for disease activity and therapy response

  • Therapeutic targeting of the AIM2 inflammasome pathway

• Cancer immunology applications:

  • Originally identified as "Absent In Melanoma 2," further exploration of its role in cancer immune surveillance

  • Potential prognostic value in various cancer types

  • Intersection with immune checkpoint pathways

• Infectious disease contexts:

  • Pathogen evasion of AIM2-mediated immunity

  • Role in bacterial, viral and parasitic infection responses

  • Potential adjuvant targets for vaccination strategies

These research directions represent emerging frontiers where AIM2 antibodies will play crucial investigative roles .

What methodological advances are improving AIM2 research capabilities?

Technological innovations are enhancing AIM2 research capabilities:

• Advanced imaging approaches:

  • Super-resolution microscopy for visualizing inflammasome assembly

  • Live-cell imaging of AIM2-DNA interactions

  • Multiplexed detection of inflammasome components

• Single-cell analytical methods:

  • Single-cell RNA-seq for heterogeneity in AIM2 expression

  • Mass cytometry for multi-parameter protein detection

  • Digital spatial profiling in tissue contexts

• Structural biology integration:

  • Antibodies for domain-specific functional studies

  • Structure-guided epitope selection

  • Cryo-EM applications for inflammasome architecture

These methodological advances are expanding the precision and scope of AIM2-focused research, opening new investigative possibilities across immunological disciplines.