ABIL2 Antibody

What is ABI2 and what cellular functions does it regulate?

ABI2 (ABL Interactor 2) is a protein encoded by the ABI2 gene located at chromosome 2q31-q33. Its structure includes a basic N-terminus with homeodomain protein homology, a central serine-rich region, 3 PEST sequences (implicated in protein degradation susceptibility), several proline-rich stretches, and an acidic C-terminus containing multiple phosphorylation sites and an SH3 domain . ABI2 functions to coordinate cytoplasmic and nuclear functions of the ABL1 tyrosine kinase . The protein has an observed molecular weight of approximately 52-56 kDa, slightly different from its calculated weight of 56 kDa, suggesting potential post-translational modifications .

Which species show confirmed reactivity with commercial ABI2 antibodies?

Current commercially available ABI2 antibodies demonstrate confirmed reactivity across human, mouse, and rat samples . This cross-species reactivity is particularly valuable for comparative studies examining ABI2 function across different mammalian models. Researchers should note that while sequence homology suggests potential reactivity with additional species, experimental validation is required before extending applications to untested organisms. When selecting an antibody for multi-species studies, those targeting highly conserved epitopes, such as the N-terminal region of ABI2, often provide the most consistent results across species .

What are the recommended applications for ABI2 antibodies?

ABI2 antibodies have been validated for multiple experimental applications with specific recommended dilutions:

Researchers should optimize these dilutions for their specific experimental conditions, as optimal concentrations may vary based on sample type and detection methods . For Western blot applications specifically, high-quality antibodies like Picoband are formulated to deliver superior quality with minimal background interference .

What are the optimal storage conditions for preserving ABI2 antibody functionality?

For maximum stability and activity retention, ABI2 antibodies should be stored at -20°C for long-term preservation (up to one year from receipt) . After reconstitution, antibodies can be stored at 4°C for up to one month, or aliquoted and returned to -20°C for extended storage (up to six months) . It is critical to avoid repeated freeze-thaw cycles, as these significantly reduce antibody activity through structural degradation .

For lyophilized antibody preparations, reconstitution should be performed with 0.2ml of distilled water to yield a concentration of 500μg/ml . Storage buffers typically contain stabilizing agents like BSA (5mg per vial) and preservatives such as Thimerosal (0.05mg) and sodium azide (0.05mg NaN3) to prevent microbial contamination and maintain antibody integrity .

How should ABI2 antibody specificity be validated in experimental systems?

A comprehensive validation strategy for ABI2 antibodies should incorporate multiple complementary approaches:

• Positive control testing: Verify antibody reactivity using samples with confirmed ABI2 expression. The following positive controls have been experimentally validated:

  • HEK-293 cells

  • Mouse heart, uterus, testis, and brain tissues

  • HeLa cells

  • K-562 cells

  • SH-SY5Y cells

• Molecular weight confirmation: The observed molecular weight of detected bands should match the expected 52-56 kDa range for ABI2 .

• Cross-reactivity assessment: Testing against related proteins, particularly other ABI family members, helps confirm specificity.

• Negative controls: Include samples lacking ABI2 expression and/or isotype control antibodies to detect non-specific binding.

• Knockdown/knockout validation: The signal should be significantly reduced or eliminated in samples where ABI2 expression has been suppressed.

For IHC applications specifically, antigen retrieval methods significantly impact results, with TE buffer (pH 9.0) recommended as the primary approach, though citrate buffer (pH 6.0) may serve as an alternative .

How can computational approaches enhance antibody design and optimization for ABI2 targeting?

Recent advances in computational antibody design offer powerful tools for researchers seeking to develop or optimize ABI2-targeted antibodies. The IsAb2.0 protocol represents a cutting-edge approach to antibody design that integrates artificial intelligence methods with traditional computational techniques . This protocol streamlines the antibody design process in several ways:

• Structure prediction: AlphaFold-Multimer (versions 2.3/3.0) generates accurate antibody-antigen complex models without requiring existing templates, enabling prediction of ABI2-antibody binding poses .

• Binding optimization: After structural modeling, tools like SnugDock can refine potential binding poses to improve accuracy .

• Hotspot identification: Computational alanine scanning predicts key antibody residues mediating ABI2 binding, providing crucial insights for affinity engineering .

• Affinity enhancement: Methods like FlexddG can identify potential single point mutations to improve binding affinity and other antibody properties .

These computational approaches significantly reduce the time and resources required for experimental antibody optimization while increasing the likelihood of successful outcomes. As demonstrated with humanized nanobody J3 (HuJ3), IsAb2.0 successfully predicted mutations that enhanced binding affinity, which were subsequently validated through experimental assays .

What strategies address non-specific binding issues when using ABI2 antibodies?

Non-specific binding represents a significant challenge in ABI2 antibody applications. Researchers can implement several evidence-based strategies to minimize this issue:

• Optimized blocking protocols: Implementing a 1-hour blocking step with 3% milk at room temperature significantly reduces non-specific binding in ELISA applications . For Western blot applications, 5% BSA in TBST often provides superior blocking compared to milk-based blockers when using phospho-specific antibodies.

• Antibody dilution optimization: Titrating antibody concentrations across a wide range (e.g., 1000 to 0.0128 nM) helps identify the optimal concentration that maximizes specific signal while minimizing background .

• Washing optimization: Implementing four washes with 0.05% PBST following primary and secondary antibody incubations effectively removes unbound antibodies .

• Detection system selection: For chemiluminescent detection, TMB (3,3′,5,5′-tetramethylbenzidine) provides high sensitivity while maintaining low background in ELISA applications .

• Cross-adsorption: Using antibodies that have been cross-adsorbed against potential cross-reactive species reduces non-specific interactions.

These approaches can be iteratively combined and optimized based on experimental outcomes to achieve maximum specificity in ABI2 detection assays.

How do post-translational modifications of ABI2 affect antibody recognition?

ABI2 undergoes several post-translational modifications (PTMs) that can significantly impact antibody recognition. The protein contains multiple phosphorylation sites within its acidic C-terminus , and phosphorylation status can alter epitope accessibility. Research indicates a discrepancy between ABI2's calculated molecular weight (55.6 kDa) and observed weight (52-56 kDa) , suggesting the presence of PTMs that affect protein migration patterns.

When investigating phosphorylation-dependent functions of ABI2, researchers should consider:

• Epitope location: Antibodies targeting regions containing phosphorylation sites may demonstrate phosphorylation-dependent recognition patterns.

• Phosphatase treatment: Comparing antibody binding before and after phosphatase treatment can reveal phosphorylation-dependent epitopes.

• Phospho-specific antibodies: For studying specific phosphorylation events, phospho-specific antibodies may be required.

• Sample preparation: Phosphatase inhibitors should be included in lysis buffers when studying phosphorylated forms of ABI2.

• Multiple detection methods: Using antibodies targeting different epitopes provides complementary data that can account for PTM-related recognition issues.

Understanding the relationship between ABI2 PTMs and antibody recognition is essential for accurate interpretation of experimental results, particularly in signaling pathway studies where phosphorylation status directly relates to protein function.

What are the most effective antigen retrieval methods for ABI2 immunohistochemistry?

Optimal antigen retrieval is critical for successful ABI2 detection in fixed tissues. Experimental validation has identified two effective methods for ABI2 immunohistochemistry:

• Primary recommended method: TE buffer at pH 9.0 has been experimentally demonstrated as the preferred antigen retrieval solution for ABI2 detection in human brain and testis tissues .

• Alternative method: Citrate buffer at pH 6.0 can serve as an alternative when TE buffer produces suboptimal results .

The effectiveness of these methods varies depending on tissue type, fixation protocol, and fixation duration. For formalin-fixed tissues with extended fixation times, the higher pH TE buffer (pH 9.0) typically provides superior epitope recovery by more effectively reversing formaldehyde-induced protein cross-linking. Researchers should systematically compare both methods when establishing protocols for new tissue types or fixation conditions.

How can researchers troubleshoot weak or absent ABI2 signals in Western blot applications?

When encountering weak or absent signals in ABI2 Western blots, researchers should systematically evaluate and optimize the following parameters:

• Antibody concentration: While recommended dilutions range from 1:2000-1:6000 for Western blot applications , higher concentrations may be necessary for samples with low ABI2 expression.

• Sample preparation: Ensure complete protein denaturation through:

  • Adequate heating (95°C for 5 minutes)

  • Appropriate reducing agent concentration

  • Sufficient SDS concentration in sample buffer

• Protein loading quantity: For tissues with low endogenous expression, increasing total protein loading (50-100 μg) may be necessary.

• Transfer efficiency: Optimize transfer conditions based on protein size:

  • For 52-56 kDa ABI2, a standard semi-dry transfer (15V for 30 minutes) or wet transfer (100V for 1 hour) typically provides efficient transfer

  • Verify transfer efficiency using reversible staining methods (Ponceau S)

• Detection sensitivity: For extremely low expression:

  • Use high-sensitivity chemiluminescent substrates

  • Extend exposure times

  • Consider signal amplification systems

If these optimizations fail to produce detectable signals, verify ABI2 expression in the sample through RT-PCR or other complementary techniques to confirm that the protein is indeed present.

What role does ABI2 play in neurodegenerative disease mechanisms?

Emerging research suggests ABI2 may have significant functions in neuronal health and neurodegenerative processes, particularly through its interactions with cytoskeletal components and signaling pathways. Recent experimental evidence demonstrates high ABI2 expression in brain tissue , with immunohistochemistry confirming specific localization patterns in human brain samples .

The structure of ABI2, with its multiple protein interaction domains including proline-rich stretches and SH3 domains , suggests extensive involvement in protein-protein interaction networks critical for neuronal function. The PEST sequences in ABI2 structure indicate regulated protein degradation , a process often dysregulated in neurodegenerative conditions.

While specific neurodegenerative disease associations continue to be investigated, ABI2's role in coordinating ABL1 tyrosine kinase functions connects it to pathways implicated in neuronal health, as ABL kinases regulate neuronal morphogenesis and synaptic function. Researchers investigating this connection should employ brain-specific positive controls, including mouse brain tissue and SH-SY5Y neuroblastoma cells, which have been validated for ABI2 detection .

How can novel AI-based approaches improve research using ABI2 antibodies?

Artificial intelligence tools are revolutionizing antibody research, with specific applications for ABI2 studies:

• Structure prediction and epitope mapping: AlphaFold-Multimer (2.3/3.0) enables accurate prediction of ABI2-antibody complex structures without requiring experimental template structures . This computational approach allows researchers to:

  • Identify optimal epitopes for antibody targeting

  • Predict cross-reactivity with related proteins

  • Design experiments based on structural insights

• Binding optimization: Advanced computational protocols like IsAb2.0 integrate multiple AI methods to enhance antibody properties :

  • FlexddG predicts mutations that improve binding affinity

  • Computational alanine scanning identifies critical binding residues

  • SnugDock refines predicted binding poses

• Experimental design optimization: AI algorithms can analyze experimental variables to identify optimal conditions for ABI2 detection, including:

  • Optimal antibody concentrations

  • Incubation time and temperature combinations

  • Buffer composition adjustments

These computational approaches significantly reduce experimental iterations, accelerate research timelines, and improve experimental outcomes. The validated success of such methods in optimizing other antibodies, such as the humanized nanobody J3 against HIV-1 gp120 , demonstrates their potential for ABI2 antibody research applications.