ABI2 Antibody

What is ABI2 protein and why is it important to study with antibodies?

ABI2 (Abl Interactor 2) is a cytoskeletal protein that plays crucial roles in cell migration, adhesion, and morphogenesis. It functions as a component of the WAVE complex, which activates actin nucleating machinery Arp2/3 to drive lamellipodia formation . ABI2 is particularly significant in neuronal development, as it controls dendritic spine morphogenesis and may promote dendritic spine specification toward large mushroom-type spines associated with memory formation . Its roles in cytoskeletal regulation make it important in cancer research, developmental biology, and neuroscience. Using antibodies against ABI2 enables researchers to study its expression patterns, subcellular localization, protein interactions, and functional roles across different experimental conditions.

How should I select the appropriate ABI2 antibody for my specific research application?

Selection of an ABI2 antibody should be based on:

• Application compatibility: Ensure the antibody is validated for your intended application (WB, IHC, etc.). For example, Proteintech's ABI2 antibody (14890-1-AP) is validated for WB (1:2000-1:6000) and IHC (1:20-1:200) .

• Species reactivity: Verify the antibody recognizes ABI2 in your experimental model. Most ABI2 antibodies react with human, mouse, and rat samples, but species-specific variations may exist .

• Antibody type: For higher specificity in applications like flow cytometry or ChIP, consider monoclonal antibodies like Abcam's EPR7069 . For broader epitope recognition in applications like Western blot, polyclonal antibodies may be preferred .

• Epitope location: When studying specific ABI2 domains or isoforms, select antibodies targeting relevant regions. For instance, Novatein's polyclonal antibody targets the N-terminus of human ABI2 (158-173aa) , while others target different regions.

• Validation data: Review the manufacturer's validation data for your specific application to ensure reliable detection of ABI2.

What are the optimal sample preparation protocols for detecting ABI2 in different applications?

For Western Blot:

• Lyse cells or tissues in RIPA buffer containing protease inhibitors

• Use 10-30 μg of total protein per lane

• Recommended running conditions: 5-20% SDS-PAGE at 70V (stacking)/90V (resolving)

• Transfer proteins to nitrocellulose membrane at 150mA for 50-90 minutes

• Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Incubate with primary antibody (0.5-1 μg/mL) overnight at 4°C

• Wash with TBS-0.1% Tween (3 times, 5 minutes each)

• Incubate with HRP-conjugated secondary antibody (1:5000) for 1.5 hours at room temperature

• Develop using enhanced chemiluminescence detection

For Immunohistochemistry:

• Use paraffin-embedded sections (5-7 μm thick)

• Perform heat-mediated antigen retrieval with citrate buffer pH 6.0 or TE buffer pH 9.0

• Block with appropriate serum (typically 5-10% normal serum)

• Incubate with primary antibody at recommended dilution (typically 1:50-1:200)

• Use appropriate detection system (e.g., HRP-polymer with DAB chromogen)

How can ABI2 antibodies be utilized to study its role in dendritic spine morphogenesis and learning/memory?

ABI2 plays a critical role in dendritic spine morphogenesis and cognitive function, as demonstrated by studies showing that ABI2-deficient mice exhibit abnormal dendritic spine morphology and density, as well as profound deficits in learning and memory . To investigate these functions:

• Co-localization studies: Use ABI2 antibodies alongside markers for dendritic spines (e.g., PSD95) in immunofluorescence to assess co-localization patterns in neuronal cultures or brain sections .

• Functional knockdown/knockout validation: ABI2 antibodies can validate successful knockdown/knockout in CRISPR-engineered or shRNA-treated neuronal models before assessing dendritic spine morphology changes .

• Spine morphology analysis: Following experimental manipulations (e.g., BDNF treatment), use ABI2 antibodies to correlate protein expression/localization with changes in spine density and morphology (mushroom, thin, or stubby spines) .

• Protein interaction studies: Use ABI2 antibodies for co-immunoprecipitation to identify interaction partners in the WAVE complex that regulates actin dynamics in dendritic spines .

• Phosphorylation state assessment: Combine ABI2 antibodies with phospho-specific antibodies to assess activity-dependent modifications that may regulate spine morphogenesis .

Research by Grove et al. demonstrated that ABI2 knockout mice showed abnormal migration in the neocortex and hippocampus, correlating with observed memory deficits, suggesting ABI2's critical role in neuronal connectivity .

What are the critical considerations when using ABI2 antibodies to study the WAVE complex regulation of actin dynamics?

The WAVE complex is essential for actin filament reorganization through interaction with the Arp2/3 complex. When studying this system with ABI2 antibodies:

• Complex integrity assessment: Use ABI2 antibodies in co-immunoprecipitation to verify the integrity of the WAVE complex and determine whether experimental manipulations disrupt the complex .

• Subcellular localization: ABI2 normally localizes to protrusive lamellipodia and filopodia tips . Use immunofluorescence with ABI2 antibodies to track changes in localization during cell migration or in response to stimuli.

• Isoform-specific effects: Be aware that isoform 1, but not isoform 3, localizes to protruding lamellipodia and filopodia tips . Select antibodies that can differentiate between isoforms if studying isoform-specific functions.

• Downstream signaling effects: When downregulating ABI2 expression (via RNAi), antibodies can confirm knockdown efficiency and correlate it with changes in WAVE complex components and lamellipodia formation .

• Temporal dynamics: For live-cell imaging studies, consider using anti-ABI2 antibodies to label the endogenous protein in fixed cells at different time points during cellular processes like migration or adhesion formation.

Grove et al. showed that downregulation of Abi expression impaired adherens junction formation and correlated with downregulation of the Wave actin-nucleation promoting factor , demonstrating the critical link between ABI2 and actin dynamics.

Why might I observe variable molecular weights for ABI2 in Western blot analysis?

ABI2 has a calculated molecular weight of approximately 56 kDa, but observed bands can range from 50-60 kDa. Potential reasons for size variability include:

• Post-translational modifications: Phosphorylation of ABI2 by ABL1/c-Abl can alter migration patterns .

• Isoform detection: Human ABI2 has multiple isoforms resulting from alternative splicing. Different antibodies may detect different isoforms based on epitope location .

• Species differences: Though highly conserved, slight differences exist between human (calculated 56 kDa) and rodent ABI2 proteins that might affect migration .

• Sample preparation: Harsh lysis conditions or heating samples above 95°C may cause protein degradation, resulting in lower molecular weight bands.

• Gel percentage: Higher percentage gels (>10%) tend to compress protein bands, potentially altering the apparent molecular weight.

To clarify which band represents true ABI2 signal, consider:

• Using positive and negative controls (e.g., ABI2 overexpression and knockdown samples)

• Running samples from multiple cell lines with known ABI2 expression (e.g., K562, HeLa, SH-SY5Y)

• Performing peptide competition assays with the immunizing peptide to identify specific bands

How can I optimize immunohistochemistry protocols to improve ABI2 detection in tissue sections?

For optimal ABI2 detection in IHC applications:

• Antigen retrieval optimization:

  • Test both citrate buffer (pH 6.0) and TE buffer (pH 9.0) antigen retrieval methods

  • Optimize retrieval time (15-30 minutes) and temperature (95-100°C)

• Antibody concentration titration:

  • Start with the manufacturer's recommended dilution range (typically 1:50-1:200 for ABI2)

  • Perform a dilution series to determine optimal signal-to-noise ratio

• Blocking optimization:

  • Test different blocking solutions (5-10% normal serum or BSA)

  • Include 0.1-0.3% Triton X-100 for improved antibody penetration in thicker sections

• Incubation conditions:

  • Compare overnight incubation at 4°C versus 1-2 hours at room temperature

  • For weak signals, consider longer primary antibody incubation or signal amplification systems

• Background reduction:

  • Include 0.1-0.3% Tween-20 in wash buffers

  • Consider adding 0.1% BSA to antibody diluent to reduce non-specific binding

  • For tissues with high endogenous peroxidase activity, use stronger blocking (3% H₂O₂ for 15-20 minutes)

The Elabscience ABI2 antibody (E-AB-18253) has been verified for IHC in human cervical cancer samples at 1:50-1:100 dilution , while Proteintech's antibody (14890-1-AP) works well in human brain and testis tissues at 1:20-1:200 .

How do polyclonal and monoclonal ABI2 antibodies compare in research applications?

Recombinant monoclonal antibodies like Abcam's EPR7069 typically show 1-2 orders of magnitude higher affinity compared to traditional mouse monoclonal antibodies , potentially offering better performance in applications requiring high sensitivity and specificity.

What are the key differences in ABI2 function between human and model organisms that might impact antibody selection?

When selecting ABI2 antibodies for cross-species studies, consider:

• Sequence homology: Human ABI2 shares high sequence homology with mouse and rat orthologs, particularly at the N-terminus region (positions 158-173) targeted by many antibodies . This enables many antibodies to cross-react across these species.

• Expression patterns:

  • Mouse: Highest expression in eye and brain tissues

  • Human: Broadly expressed but significant in neural tissues

  • Both species show similar subcellular localization patterns in lamellipodia and filopodia

• Functional conservation:

  • The role of ABI2 in actin cytoskeleton regulation via the WAVE complex is conserved across mammals

  • Mouse knockout studies show phenotypes in lens fiber migration, neuronal migration, and memory formation that are likely relevant to human biology

• Species-specific antibody validation:

  • Boster Bio's PA2068 antibody has been validated in both human cell lines (293T, HeLa, K562, SH-SY5Y) and rodent samples (rat brain, rat PC-12, mouse brain)

  • Proteintech's 14890-1-AP shows reactivity with human, mouse, and rat samples

  • Some antibodies may show species-specific differences in optimal working dilutions

• Molecular weight differences:

  • Human ABI2: Calculated 56 kDa, observed 52-60 kDa

  • Mouse ABI2: Similar but may show slight variations in migration patterns

When studying feline ABI2, researchers should note that while specific validation may be lacking, there's likely cross-reactivity based on sequence conservation, as suggested by manufacturer responses to customer inquiries .

How is ABI2 research contributing to our understanding of neurological disorders and potential therapeutic targets?

Recent ABI2 research has significant implications for neurological disorders:

• Learning and memory deficits: ABI2-deficient mice exhibit profound deficits in short- and long-term memory, suggesting ABI2's importance in cognitive function . This makes ABI2 signaling pathways potential targets for cognitive enhancement therapies.

• Dendritic spine abnormalities: Given ABI2's role in dendritic spine morphogenesis, its dysfunction may contribute to conditions characterized by spine abnormalities, including autism spectrum disorders, intellectual disability, and schizophrenia .

• Neuronal migration disorders: ABI2-knockout mice show defective neuronal migration in the neocortex and hippocampus , suggesting potential involvement in human neurodevelopmental disorders associated with neuronal migration defects.

• Synaptic plasticity: ABI2's involvement in actin dynamics at dendritic spines suggests it may play a role in synaptic plasticity mechanisms underlying learning and memory . Antibodies against ABI2 are valuable tools for studying these processes.

• BDNF-TrkB signaling: ABI2 may mediate actin-dependent BDNF-NTRK2 early endocytic trafficking that triggers dendrite outgrowth , linking it to neurotrophin signaling pathways implicated in multiple neurological disorders.

Researchers can use ABI2 antibodies to investigate these pathways in patient-derived samples or disease models, potentially identifying novel therapeutic approaches targeting actin cytoskeleton regulation.

What new methodological approaches are enhancing ABI2 antibody applications in cutting-edge research?

Emerging methodologies are expanding ABI2 antibody applications:

• Super-resolution microscopy: Beyond conventional immunofluorescence, techniques like STED, STORM, and PALM enable visualization of ABI2 localization in dendritic spines and lamellipodia with nanometer resolution, revealing previously undetectable details of its organization within the WAVE complex .

• Proximity ligation assays (PLA): This technique allows detection of protein-protein interactions between ABI2 and its binding partners in situ, providing spatial information about where in the cell these interactions occur - particularly valuable for studying ABI2's interactions with ABL kinases and WAVE complex components .

• Live-cell imaging with genetically-encoded tags: Combining antibody validation with fluorescently-tagged ABI2 enables real-time monitoring of protein dynamics during processes like lamellipodia formation and adherens junction assembly .

• Multi-omics integration: Correlating ABI2 antibody-based proteomics with transcriptomics and functional genomics provides comprehensive understanding of ABI2's role in signaling networks, as demonstrated in studies of ABI2-deficient mouse models .

• CRISPR-edited cellular models: ABI2 antibodies are essential for validating CRISPR-engineered cellular models, such as the ABI2-CRISPR lines developed to study ABI2's role in flowering regulation in plants , providing templates for similar approaches in mammalian systems.

• Patient-derived organoids: ABI2 antibodies can be applied to study protein expression and localization in 3D organoid models derived from patient cells, offering insights into disease-specific alterations in ABI2 function in a physiologically relevant context.

These advanced methods, coupled with highly specific ABI2 antibodies, are enabling unprecedented insights into ABI2's molecular functions and potential therapeutic applications.