ABL2 Antibody

What is ABL2 and why is it significant in research?

ABL2 (also known as ARG) is a non-receptor tyrosine kinase that regulates cytoskeleton remodeling, cell motility, adhesion, and receptor endocytosis. It coordinates actin remodeling through tyrosine phosphorylation of proteins controlling cytoskeleton dynamics such as MYH10, CTTN, TUBA1, and TUBB. ABL2 binds directly to F-actin and regulates actin cytoskeletal structure through its F-actin-bundling activity .

In research, ABL2 is significant for:

• Understanding cytoskeletal regulation mechanisms

• Studying cellular migration and invasion

• Investigating synaptic plasticity in neurons

• Examining pathogen interactions with host cytoskeleton

• Exploring muscle development and fiber length regulation

• Cancer research, particularly in hepatocellular carcinoma

ABL2 is highly expressed in proliferating myoblasts and declines during differentiation, suggesting a role in regulating myogenesis .

What types of ABL2 antibodies are available and how do they differ?

Various types of ABL2 antibodies are available for research applications:

Key differences include:

• Polyclonal antibodies: Recognize multiple epitopes, potentially providing stronger signals but with possible cross-reactivity

• Monoclonal antibodies: Higher specificity for single epitopes with potentially lower background

• Recombinant antibodies: Offer batch-to-batch consistency compared to traditional antibodies

• Dual specificity antibodies: Detect both ABL1 and ABL2, useful for studying related functions

How should I design experiments to study ABL2 expression across different cell types or tissues?

When designing experiments to study ABL2 expression patterns:

Methodological approach:

• Choose appropriate antibodies: Select antibodies validated for your specific application and species of interest. For example, rabbit polyclonal antibodies (17693-1-AP) have been validated for WB in mouse brain tissue, IP in HeLa cells, and IHC in human gliomas tissue .

• Include proper controls:

  • Positive control: Mouse brain tissue for Western blot

  • Negative control: ABL2 knockout or knockdown tissues/cells

  • Loading controls: Pan-Cadherin for whole cell lysates

• Expression analysis methods:

  • Western blotting: Use 1:1000-1:4000 dilution of anti-ABL2 antibody

  • Immunohistochemistry: Use 1:50-1:500 dilution with appropriate antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • In situ hybridization: For embryonic tissues, use digoxigenin-labeled riboprobes directed against mRNA encoding 1088 bp and 1321 bp fragments of ABL2

  • Immunoprecipitation: Use 0.5-4.0 μg antibody for 1.0-3.0 mg total protein lysate

• Time course studies: For differentiation studies, collect cells at sequential timepoints. Research shows ABL2 is highly expressed in proliferating myoblasts (C2C12 cells in growth medium) and declines during differentiation .

What are the optimal conditions for Western blotting with ABL2 antibodies?

Detailed Western blotting protocol for ABL2 detection:

• Lysate preparation:

  • Lyse cells in buffer containing: 30 mM triethanolamine, 1% NP-40, 50 mM NaF, 2 mM NaV₂O₅, 1 mM Na-tetrathionate, 5 mM EDTA, 5 mM EGTA, 100 mM N-ethylmaleimide, 50 mM NaCl, and protease inhibitors

  • Incubate for 30 min at 4°C

  • Centrifuge at 12,000 rpm for 20 min at 4°C to remove debris

  • Measure protein concentration by Bradford assay

• Gel electrophoresis:

  • Load 50 μg of protein lysate

  • Use 7.5% or 4-12% gradient gels for optimal resolution of ABL2 (128-140 kDa)

• Antibody incubation:

  • Primary antibody: Anti-ABL2 (17693-1-AP) at 1:1000-1:4000 dilution

  • Incubation time: Overnight at 4°C or room temperature for 1.5 hours

• Detection notes:

  • ABL2 typically appears as three distinct bands between 100-150 kDa in C2C12 cells in growth media

  • Expected molecular weight is 128-140 kDa depending on isoform

  • Use enhanced chemiluminescence for detection

• Loading controls:

  • Pan-Cadherin for membrane fractions

  • Myosin heavy chain (My32) for muscle cells

How can ABL2 antibodies be used to investigate ABL2's role in cytoskeletal regulation?

ABL2 regulates cytoskeletal dynamics through multiple mechanisms. To investigate these functions:

Methodological approaches:

• Co-immunoprecipitation studies:

  • Use ABL2 antibodies to immunoprecipitate ABL2 and its interacting partners

  • Western blot for known cytoskeletal regulators (CTTN, MYH10, TUBA1, TUBB)

  • This reveals ABL2's physical interactions with cytoskeletal components

• Phosphorylation analysis:

  • After ABL2 knockdown, analyze phosphorylation status of downstream targets

  • Example: In hepatocellular carcinoma studies, researchers examined phospho-cortactin (Tyr466) levels after ABL2 siRNA treatment

  • This reveals ABL2's kinase activity on cytoskeletal regulators

• Cell migration/invasion assays with ABL2 manipulation:

  • Transfect cells with ABL2 siRNA (validated in HepG2 cells)

  • Perform transwell migration and invasion assays

  • Compare migration rates between ABL2-knockdown cells and controls

  • Research shows ABL2 silencing significantly impacts migration capabilities

• Visualization of cytoskeletal structures:

  • Use immunofluorescence with ABL2 antibodies (1:200-1:1000 dilution)

  • Co-stain with actin markers (phalloidin) or microtubule markers

  • This reveals co-localization of ABL2 with cytoskeletal elements

What are the considerations when using ABL2 antibodies in immunohistochemistry for cancer research?

When using ABL2 antibodies for IHC in cancer tissues:

Technical considerations:

• Antibody selection and validation:

  • Use antibodies validated for IHC in the specific cancer type

  • Example: Anti-ABL2 17693-1-AP has been validated for human gliomas tissue

• Antigen retrieval optimization:

  • Primary option: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Inadequate antigen retrieval is a common cause of false negatives

• Scoring and quantification method:

  • Define clear criteria for positive staining

  • Example from HCC research: "Immunostaining was defined as high expression level if moderate or strong staining was observed in more than 10% of the cancer cells"

• Controls:

  • Positive control: Known ABL2-expressing tissue

  • Negative control: Non-specific mouse/rabbit IgG

  • Include normal adjacent tissue for comparison

• Correlation with clinical parameters:

  • Research shows ABL2 overexpression predicts poor prognosis in hepatocellular carcinoma

  • Document patient characteristics, tumor stage, and survival data for correlation analysis

What are common issues with ABL2 Western blotting and how can they be addressed?

Common issues and solutions:

How do I interpret ABL2 expression data in relation to cell differentiation status?

Methodological approach to interpretation:

• Baseline expression understanding:

  • ABL2 is highly expressed in proliferating myoblasts

  • Expression declines during differentiation

  • In C2C12 cells, ABL2 migrates as three distinct bands between 100-150 kDa in growth media

• Quantification method:

  • Normalize ABL2 expression to a loading control

  • Express values relative to growth medium condition (assign GM a value of 1.0)

  • Plot expression over differentiation time course

• Expression pattern analysis:

  • Compare expression in:

    • Growth medium (proliferating cells)

    • Differentiation medium (days 1-7)

  • Research shows significant decrease in ABL2 expression as differentiation progresses

• Functional correlation:

  • ABL2 expression correlates with myoblast proliferation status

  • To confirm functional relevance, use conditional knockout approaches targeting specific developmental stages:

    • Pre-migratory myogenic precursors (Pax3-Cre)

    • Committed myoblasts (Myod-Cre)

    • Multinucleated myofibers (Mck-Cre)

How can ABL2 antibodies be used to study the germline bias in antibody sequences?

This question explores the intersection between ABL2 research and antibody technology development.

Methodological approach:

• Understanding germline bias:
  Research shows that antibody sequences have a germline bias, with most residues being germline-derived. Even highly matured antibodies typically have only 15-20 non-germline (NGL) residues outside their CDR3s across both chains .

• Using ABL2 as a model antigen:

  • Generate and characterize anti-ABL2 antibodies using different approaches

  • Compare conventionally-derived antibodies with those designed to overcome germline bias

  • Analyze binding characteristics, specificity, and performance in various applications

• Evaluation metrics:

  • Specificity for ABL2 vs. related ABL1

  • Ability to recognize different ABL2 isoforms

  • Performance across multiple applications (WB, IHC, IP)

  • Ability to detect ABL2 in different conformation states

• Application in designing improved research tools:

  • Understanding germline bias can help develop better antibody-specific language models (LMs) like AbLang-2

  • These models can suggest diverse sets of valid mutations with high cumulative probability

  • This approach can potentially improve ABL2 antibody specificity and performance

How can I design experiments to study ABL2's role in neurotransmission?

ABL2 may regulate neurotransmission by phosphorylating proteins at the synapse. Research approaches include:

Experimental design strategy:

• Electrophysiological assessment:

  • Compare AMPA and NMDA receptor current densities between control and ABL2 knockdown neurons

  • Research shows ABL2 knockdown increases AMPA current density by ~29% and NMDA current density by ~48%

  • Perform patch-clamp recordings after transfecting neurons with siRNA against ABL2

• Molecular manipulation approaches:

  • siRNA knockdown: Use validated siRNA sequences to reduce ABL2 expression

  • Lentiviral overexpression: Generate lentiviruses expressing:

    • Wild-type ABL2 (Abl2-WT)

    • Kinase-dead mutants (Abl2-KD)

    • Empty vector controls

  • Infect primary neuronal cultures on days 3-5 in vitro

• Synaptic protein analysis:

  • Examine phosphorylation status of synaptic proteins in the presence or absence of ABL2

  • Perform co-immunoprecipitation to identify ABL2 interacting partners at the synapse

  • Analyze postsynaptic density fractions for ABL2 localization

• Statistical analysis:

  • Use appropriate statistical tests (e.g., Kruskal-Willis One-way ANOVA with post hoc Dunn's analysis for non-parametric data)

  • Compare multiple conditions: siControl vs. siAbl1 vs. siAbl2

  • Establish significance thresholds (p < 0.05, 0.01, or 0.001)