AIP1-2 Antibody

What is AIP1-2 and what cellular functions does it regulate?

AIP1-2 (ASK1-Interacting Protein 1-2) is a signaling scaffold protein that interacts with actin and modulates several pathways associated with inflammation, apoptosis, and cellular development. In plants such as Arabidopsis, AIP1-2 specifically binds to actin isoforms including ACT2 and ACT7, regulating cytoskeletal organization . In mammalian systems, AIP1 functions in TNF-α-induced ASK1 activation by facilitating dissociation of inhibitory proteins like 14-3-3, thereby initiating apoptotic signaling pathways . Additionally, AIP1 modulates several MAPK signaling pathways including JNK, p38 MAPK, and ERK1/2, which are critical for inflammatory responses and insulin sensitivity .

For optimal Western blot detection using AIP1-2 antibodies:

• Sample preparation: Extract proteins using RIPA buffer with protease and phosphatase inhibitors to preserve protein integrity .

• Protein loading: Load 20-30 μg of total protein per lane for cell lysates; higher amounts (50-75 μg) may be needed for tissue extracts .

• Gel conditions: Use 7.5% SDS-PAGE for optimal separation, as AIP1 has a molecular weight around 130 kDa .

• Transfer conditions: Transfer to PVDF membrane at 100V for 90 minutes in standard transfer buffer with 20% methanol.

• Blocking: Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Dilute anti-AIP1-2 antibody typically 1:500-1:1000 in blocking buffer; incubate overnight at 4°C.

• Secondary antibody: Use appropriate HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature.

• Detection: Visualize using ECL substrates with exposure times of 1-5 minutes depending on signal strength .

Expected band size for AIP1 is approximately 130 kDa, though specific isoforms may vary slightly in molecular weight .

How can I distinguish between AIP1 isoforms in experimental systems?

Distinguishing between AIP1 isoforms requires careful experimental design:

• Isoform-specific antibodies: Use antibodies raised against unique epitopes. For example, antibodies targeting the C-terminal region (aa 1350 to C-terminus for MAGI2/AIP1) can provide isoform specificity .

• RT-PCR approach: Design primers that span unique exon-exon junctions or target isoform-specific sequences. For AIP1-2, semi-quantitative RT-PCR can confirm expression as demonstrated in Arabidopsis studies with primers flanking the insertion sites in the fifth exon (aip1.2-1) or fifth intron (aip1.2-2) .

• Complementation assays: Re-express specific isoforms in knockout cells to confirm isoform-specific functions. This approach successfully identified the unique roles of AIP1-2 in Arabidopsis development when combined with act7 mutations .

What controls are critical for validating AIP1-2 antibody specificity?

Rigorous validation of AIP1-2 antibody specificity requires multiple controls:

• Genetic knockout/knockdown validation: Compare antibody signal in wild-type versus AIP1-2 knockout/knockdown samples. In research with Arabidopsis, T-DNA insertion mutants (aip1.2-1 and aip1.2-2) provided essential negative controls for antibody validation .

• Recombinant protein controls: Use purified GST-AIP1-2 or His-tagged AIP1-2 as positive controls in immunoblotting. These recombinant proteins have been successfully used in pull-down assays to verify interactions with actin .

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide prior to immunostaining or Western blotting. Signal reduction confirms specificity for the target epitope.

• Cross-reactivity assessment: Test the antibody against related proteins (e.g., AIP1-1 in plant systems) to ensure specificity for the desired isoform .

• Multiple antibody validation: Compare results using antibodies targeting different epitopes of the same protein. This approach helps confirm signal specificity across different experimental conditions.

• RNAi rescue experiment: Restore protein expression using RNAi-resistant constructs to validate antibody-detected phenotypes. The RNase protection assay with AIP1 shRNA has been used to confirm specific silencing .

How should I design experiments to study AIP1-2 interactions with signaling proteins?

To effectively study AIP1-2 interactions with signaling proteins:

• Co-immunoprecipitation (Co-IP):

  • Use anti-AIP1 antibody for immunoprecipitation followed by immunoblotting with antibodies against potential interacting partners (e.g., ASK1, 14-3-3, actins)

  • Include appropriate detergent conditions (typically 0.5-1% NP-40 or Triton X-100) to maintain protein-protein interactions

  • Validate with reverse Co-IP (precipitate with partner antibody, blot for AIP1)

• Pull-down assays:

  • Express GST-AIP1-2 in bacterial systems for affinity purification

  • Incubate with cell/tissue lysates to capture interacting proteins

  • This approach successfully demonstrated AIP1-2 interactions with actin isoforms (ACT2, ACT7) in Arabidopsis

• Proximity ligation assay (PLA):

  • Use for in situ detection of protein-protein interactions in fixed cells

  • Requires specific primary antibodies against both AIP1-2 and its interaction partner

  • Provides spatial information about interaction sites within cells

• Mutational analysis:

  • Generate AIP1-2 constructs with specific domain deletions or point mutations

  • Test the effect on protein interactions and downstream signaling

  • This approach can map specific interaction domains (e.g., how the PH and C2 domains of AIP1 mediate protein binding)

• Functional validation:

  • After identifying interactions, validate functional relevance using genetic approaches

  • The synergistic phenotypes observed in act7;aip1-2 double mutants provide strong evidence for functional interaction between these proteins

How is AIP1-2 expression altered in pathological conditions, and how can antibodies help study these changes?

AIP1 expression changes have been documented in several pathological conditions:

• Type 2 Diabetes:

  • AIP1 is significantly downregulated in omental adipose tissue of obese patients with T2D

  • AIP1 levels negatively correlate with insulin resistance markers (HOMA-IR, r = -0.4829) and waist-to-hip ratio (r = -0.2614)

  • Anti-AIP1 antibodies can quantify expression changes via Western blot and IHC in adipose tissue samples

  • After bariatric surgery (RYGB), AIP1 expression in adipose tissue normalizes, correlating with metabolic improvement

• Alzheimer's Disease:

  • AIP1 is elevated in brain tissue from AD Tg2576 mice at both mRNA and protein levels

  • Aβ1-42 treatment increases AIP1 expression in cerebral microvascular endothelial cells

  • Antibodies against AIP1 can track these expression changes in brain tissue sections and primary cell cultures via Western blot and immunohistochemistry

• Inflammatory Conditions:

  • AIP1 modulates TNF-α-induced inflammatory signaling

  • Antibodies against phosphorylated downstream targets (p-JNK, p-p38, p-ERK1/2) help evaluate the functional consequences of AIP1 dysregulation

When studying these changes, it's critical to use consistent sample collection protocols, appropriate normalization controls, and validated antibody dilutions for quantitative comparisons across disease stages.

What methodological considerations are important when using AIP1-2 antibodies in tissue section analysis?

For optimal immunohistochemical analysis of AIP1-2 in tissue sections:

• Tissue fixation and processing:

  • Fix tissues in 4% paraformaldehyde for 24h

  • Paraffin-embed and cut into 5-μm-thick sections

  • For AIP1 detection in adipose tissue, special handling may be required to preserve tissue architecture

• Antigen retrieval:

  • Use Citrate Antigen Retrieval solution at 95°C for 20 minutes

  • Cool to room temperature before proceeding with staining

  • This step is critical for exposing AIP1 epitopes that may be masked during fixation

• Blocking and background reduction:

  • Block endogenous peroxidase with 3% H₂O₂ in methanol for 12 minutes

  • Block nonspecific binding with 10% goat serum for 1 hour

  • Include rabbit IgG antibody at the same concentration as the primary antibody as a negative control

• Antibody concentrations and incubation:

  • For AIP1 detection, use rabbit anti-AIP1 antibody at 0.5 μg/ml

  • For co-staining with inflammatory markers (e.g., TNF-α), use rabbit anti-TNF-α at 2.5 μg/ml

  • Incubate primary antibodies overnight at 4°C to maximize specific binding

• Visualization and counterstaining:

  • DAB staining for antibody detection followed by hematoxylin counterstaining for nuclei

  • Capture images using standardized microscopy settings to enable quantitative comparisons

How can I integrate AIP1-2 antibodies into multiplex protein analysis workflows?

To effectively integrate AIP1-2 antibodies into multiplex protein analysis:

• Multiplex immunofluorescence:

  • Use spectrally distinct fluorophores for simultaneous detection of AIP1-2 and interacting proteins

  • For example, co-staining of AIP1 with phosphorylated ASK1 (pSer-967) and 14-3-3 can visualize the dynamic regulation of this signaling complex

  • Select antibodies raised in different host species to avoid cross-reactivity with secondary antibodies

  • Include single-stain controls to establish specificity and assess bleed-through

• Protein array applications:

  • Use purified AIP1-2 antibodies in reverse-phase protein arrays to screen multiple samples simultaneously

  • Include appropriate controls for normalization and antibody validation

  • This approach is valuable for screening AIP1-2 expression across multiple patient samples or experimental conditions

• Mass cytometry (CyTOF):

  • Label AIP1-2 antibodies with metal isotopes for inclusion in high-dimensional CyTOF panels

  • Enables simultaneous measurement of AIP1-2 with dozens of other cellular markers

  • Particularly useful for analyzing AIP1-2 expression in heterogeneous cell populations

• Proximity extension assay:

  • Utilize antibody pairs recognizing different epitopes on AIP1-2

  • Conjugate with DNA oligonucleotides that can form amplifiable reporters when in close proximity

  • Enables highly sensitive detection of AIP1-2 in complex biological samples

• Implementation considerations:

  • Validate antibody performance in multiplexed format against single-plex controls

  • Optimize antibody concentrations to achieve comparable sensitivity across targets

  • Include both technical and biological replicates to ensure reproducibility

What are common issues in AIP1-2 Western blotting and how can they be resolved?

Common Western blot challenges with AIP1-2 antibodies include:

For optimal AIP1-2 detection, SDS-PAGE conditions should be adjusted for its molecular weight (approximately 130 kDa), and 7.5% gels are recommended for better resolution in this size range .

How can I optimize immunoprecipitation protocols for studying AIP1-2 interactions?

To optimize immunoprecipitation (IP) protocols for AIP1-2:

• Lysis buffer optimization:

  • Use buffers that preserve protein-protein interactions while efficiently extracting AIP1-2

  • For co-IP of AIP1-2 with ASK1, a buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 1% Triton X-100, 10% glycerol, and protease/phosphatase inhibitors works effectively

  • Avoid harsh detergents like SDS that disrupt protein-protein interactions

• Antibody selection and coupling:

  • For AIP1 IP, anti-AIP1 antibodies should be carefully validated for specificity

  • Pre-couple antibodies to Protein A/G beads (2-5 μg antibody per 20 μl beads) for more efficient capture

  • For difficult interactions, consider crosslinking antibodies to beads using dimethyl pimelimidate (DMP)

• Pre-clearing samples:

  • Pre-clear lysates with Protein A/G beads for 1 hour at 4°C before adding antibody

  • This reduces nonspecific binding and decreases background

• IP conditions optimization:

  • Perform IP overnight at 4°C with gentle rotation to maximize specific interactions

  • After IP, wash beads 3-5 times with lysis buffer to remove nonspecific binding

  • For detecting transient interactions, consider shorter incubation times or chemical crosslinking

• Elution conditions:

  • For Western blot analysis, elute in Laemmli buffer at 70°C (not boiling) for 10 minutes

  • For mass spectrometry analysis, consider gentler elution using competing peptides or low pH

This approach has successfully detected interactions between AIP1 and ASK1, as well as the dissociation of 14-3-3 from ASK1 in response to Aβ1-42 treatment .

When should I consider using monoclonal versus polyclonal antibodies for AIP1-2 research?

The choice between monoclonal and polyclonal antibodies for AIP1-2 research depends on specific experimental goals:

Use monoclonal antibodies when:

• High reproducibility between experiments is critical

• Specific epitopes or post-translational modifications need to be detected

• Background must be minimized for clean Western blots or immunostaining

• Long-term studies requiring consistent antibody performance are planned

• Applications involve flow cytometry or immunoprecipitation of specific AIP1-2 forms

Use polyclonal antibodies when:

• Maximum sensitivity is needed (multiple epitopes enhance signal)

• Detecting AIP1-2 across multiple species (broader epitope recognition)

• Studying native protein conformation or protein complexes

• Performing initial characterization before investing in monoclonals

• Working with fixed tissues where epitope availability may be limited

Research-based recommendations:

• For studying specific AIP1-2 isoforms, monoclonal antibodies against unique regions provide better discrimination

• For detecting endogenous AIP1-2 in tissues, polyclonal antibodies like those used in adipose tissue studies show good sensitivity

• For co-immunoprecipitation experiments studying AIP1-ASK1 interactions, polyclonal antibodies have been successfully used

• When studying signaling dynamics, monoclonal phospho-specific antibodies against downstream targets provide clearer results

The rabbit polyclonal antibody against AIP1 has demonstrated successful application in Western blot, immunohistochemistry, and co-immunoprecipitation experiments .

How can AIP1-2 antibodies be utilized in engineered therapeutic systems?

AIP1-2 antibodies show potential in engineered therapeutic applications:

• Synthetic cytokine receptor systems:

  • Anti-idiotypic nanobodies (AIP VHH) against palivizumab have been developed as components of synthetic cytokine receptors

  • These systems utilize engineered IgG2 subclass antibodies to activate designer receptors

  • The AIP2gp130Δstalk construct demonstrates how AIP components can be incorporated into functional signaling systems

  • This approach allows for controlled activation of signaling pathways in therapeutic cell engineering

• Monitoring therapeutic response:

  • AIP1 expression normalizes after bariatric surgery in T2D patients, making it a potential biomarker for treatment efficacy

  • Antibodies against AIP1 can quantify these changes in adipose tissue biopsies

  • Changes in AIP1 levels correlate with improvements in clinical metrics including HOMA-IR

• Target validation for drug development:

  • AIP1-ASK1-JNK signaling represents a potential therapeutic axis in multiple diseases

  • In Alzheimer's disease models, AIP1 silencing attenuates Aβ1-42-induced apoptosis in cerebral endothelial cells

  • AIP1-2 antibodies provide essential tools for validating this pathway as a drug target

• Cell-based therapy monitoring:

  • In engineered T-cell systems, AIP-based synthetic receptors can be monitored using specific antibodies

  • Flow cytometric analysis with anti-AIP antibodies can confirm successful expression of synthetic receptors on T-cell surfaces

What novel protocols combine AIP1-2 antibodies with emerging technologies?

Emerging technologies are expanding AIP1-2 antibody applications:

• Single-cell protein analysis:

  • Integration of AIP1-2 antibodies into single-cell Western blot platforms

  • Allows correlation of AIP1-2 expression with cellular heterogeneity

  • Particularly valuable for analyzing AIP1-2 expression in mixed cell populations from tissue samples

• Spatial transcriptomics with protein correlation:

  • Combining anti-AIP1-2 immunofluorescence with spatial transcriptomics

  • Reveals relationships between AIP1-2 protein expression and spatial gene expression patterns

  • Critical for understanding AIP1-2 function in complex tissues like adipose depots or brain sections

• CRISPR screening validation:

  • Using AIP1-2 antibodies to validate CRISPR knockout/knockin efficiency

  • The documented sgRNA sequences (5'-3': caccgAAGTACCTGCAGGACGCCCT) provide starting points for CRISPR-based AIP1 manipulation

  • Antibodies confirm protein-level changes following genetic modification

• Living cell imaging of AIP1-2 dynamics:

  • Anti-AIP1-2 nanobodies conjugated to fluorescent proteins for live-cell imaging

  • Enables real-time visualization of AIP1-2 translocation and complex formation

  • Critical for understanding the temporal dynamics of AIP1-ASK1-14-3-3 interactions

• Mass spectrometry integration:

  • Immunoprecipitation with AIP1-2 antibodies followed by mass spectrometry

  • Identifies novel interaction partners and post-translational modifications

  • Provides comprehensive understanding of AIP1-2 in signaling networks

What are current challenges in reproducibility with AIP1-2 antibodies and how can they be addressed?

Key challenges in AIP1-2 antibody reproducibility include:

• Epitope specificity issues:

  • AIP1 contains multiple domains (PH, C2, GAP, others) that can generate different antibody epitopes

  • Solution: Clearly document epitope information (e.g., "Synthetic Peptide within Human MAGI2 aa 1350 to C-terminus" ) and validate with recombinant domain proteins

• Isoform cross-reactivity:

  • Multiple AIP1 isoforms (AIP1-1, AIP1-2) may cross-react with antibodies

  • Solution: Validate antibody specificity using recombinant AIP1 isoforms and knockout controls for each specific isoform

• Species-specific validation gaps:

  • Many antibodies are validated in limited species (often human only)

  • Solution: Perform cross-species validation using sequence alignment and recombinant proteins from target species

• Application-specific optimization:

  • Antibodies may work in Western blot but fail in immunohistochemistry

  • Solution: Validate each antibody for specific applications rather than assuming cross-application functionality

• Standardization of protocols:

  • Variable results between labs due to protocol differences

  • Solution: Adopt detailed standardized protocols for sample preparation, antibody dilutions, and detection methods

• Reproducibility documentation:

  • Inadequate reporting of antibody validation data

  • Solution: Include complete validation data including lot numbers, dilutions, positive and negative controls, and full immunoblot images in publications