Phospho-ABL1 (Y245) Antibody

What is the significance of ABL1 phosphorylation at Y245?

The phosphorylation of ABL1 at tyrosine 245 is a key regulatory event that activates this tyrosine kinase and its downstream signaling pathways. This specific phosphorylation site plays a crucial role in modulating ABL1's activity, which influences cellular processes including cell growth, differentiation, and survival . In research contexts, detecting phosphorylation at this site allows scientists to monitor ABL1 activation status, which is particularly relevant in cancer research and when studying cellular response to various stimuli.

What applications are Phospho-ABL1 (Y245) antibodies validated for?

Phospho-ABL1 (Y245) antibodies are validated for multiple applications across different research platforms:

• Western Blot (WB): Typically at dilutions of 1:500-1:2000

• Immunohistochemistry (IHC-P): Usually at dilutions of 1:50-1:200

• Immunofluorescence (IF): Generally at dilutions of 1:200-1:1000

• Immunocytochemistry (ICC): At similar dilutions to IF

• ELISA: Often at higher dilutions of approximately 1:5000

The specific dilution requirements may vary between manufacturers and should be optimized for your specific experimental conditions.

Which species reactivity is available for Phospho-ABL1 (Y245) antibodies?

Most commercially available Phospho-ABL1 (Y245) antibodies demonstrate cross-reactivity with multiple species:

When selecting an antibody for your research, confirm the reactivity with your species of interest and consider validating the antibody in your specific model system before proceeding with full experiments .

What is the typical storage and handling protocol for these antibodies?

For optimal antibody performance, follow these storage guidelines:

• Long-term storage: -20°C for up to one year

• Short-term/frequent use: 4°C for up to one month

• Avoid repeated freeze-thaw cycles as they may compromise antibody integrity

• Many antibodies are supplied in PBS with additives such as glycerol (50%), BSA (0.5%), and sodium azide (0.02%) to maintain stability

Aliquoting the antibody upon receipt can minimize freeze-thaw cycles and extend the antibody's useful life .

How can I validate the specificity of a Phospho-ABL1 (Y245) antibody in my experimental system?

Validation of phospho-specific antibodies requires careful experimental design:

• Positive and negative controls:

  • Treat cells with phosphatase inhibitors like pervanadate to generate positive controls with increased phosphorylation levels

  • Compare treated vs. untreated samples (e.g., Jurkat cells with/without pervanadate)

  • Use lambda phosphatase treatment as a negative control to remove phosphorylation

• Multiple detection methods:

  • Confirm results using both Western blot and immunofluorescence

  • Serial dilutions of cell lysates in ELISA to establish detection limits

• Knockdown/knockout validation:

  • siRNA knockdown of ABL1 should reduce or eliminate the specific signal

  • CRISPR/Cas9 knockout cells provide definitive negative controls

• Peptide competition assay:

  • Pre-incubate antibody with the phosphorylated peptide immunogen

  • Signal should be significantly reduced or eliminated

Troubleshooting tip: If background signal is high, optimize blocking conditions and increase washing steps. Consider testing different antibody dilutions to determine the optimal signal-to-noise ratio .

How do phosphorylation patterns of ABL1-Y245 differ between normal and cancer cell models?

Phosphorylation of ABL1 at Y245 shows distinct patterns between normal and cancer cells:

• Normal cells:

  • Tightly regulated, transient phosphorylation in response to specific stimuli

  • Primarily nuclear and cytoplasmic localization

  • Lower basal levels of phosphorylation

• Cancer cells (particularly leukemia):

  • Elevated constitutive phosphorylation

  • Altered subcellular localization

  • In BCR-ABL1 fusion-positive cells (e.g., K562), distinctive localization patterns compared to cells with wild-type ABL1

Research approach: Use confocal laser scanning microscopy with dual immunofluorescence labeling using anti-total ABL1 (green fluorochrome) and anti-phospho-ABL1-Y245 (red fluorochrome) with DAPI nuclear counterstaining. This allows visualization of both total protein distribution and the specific phosphorylated fraction .

Quantitative analysis should include:

• Ratio of phosphorylated to total ABL1

• Subcellular distribution analysis

• Colocalization with other signaling molecules

What are the methodological differences between detecting phospho-ABL1 (Y245) and total ABL1 in multiplexed assays?

Developing successful multiplexed detection assays requires careful consideration of several factors:

• Antibody compatibility:

  • Ensure antibodies for phospho and total protein do not cross-react

  • If using same species antibodies, consider directly conjugated antibodies

  • Sequential staining protocols may be necessary to avoid cross-reactivity

• Epitope accessibility:

  • Phosphorylation may alter protein conformation, affecting epitope availability

  • Different extraction/fixation methods may be required for optimal detection of both forms

• Quantification approaches:

  • Normalize phospho-ABL1 signal to total ABL1 for accurate activation assessment

  • Use specialized ELISA kits designed for simultaneous detection

  • For Western blotting, strip and reprobe membranes or use dual-color detection systems

• Sample preparation considerations:

  • Phosphatase inhibitors are critical in all buffers

  • Sample processing time should be minimized

  • Solubilization at appropriate cell concentrations (e.g., 4 × 10^7 cells/ml) in specialized cell lysate buffers

Recommended validation: Always confirm multiplexed results with single-target detection methods initially to ensure no interference between detection systems.

How can I interpret conflicting phospho-ABL1 (Y245) results across different detection methods?

When facing discrepancies between detection methods, consider these analytical approaches:

• Method-specific limitations:

  • Western blot: Higher sensitivity but less spatial information

  • Immunofluorescence: Provides spatial context but potential for fixation artifacts

  • ELISA: Quantitative but may miss compartmentalization details

  • IHC: Tissue context but potential antigen masking issues

• Systematic troubleshooting protocol:

  • Verify antibody lot consistency across experiments

  • Standardize lysate preparation with strict adherence to phosphatase inhibitor usage

  • Confirm protein loading normalization methods are consistent

  • Check cell treatment conditions (timing, concentration gradients)

• Biological interpretation framework:

  • Transient phosphorylation events may be captured differently by various methods

  • Consider phosphorylation kinetics and stability

  • Evaluate subcellular fractionation results separately from whole-cell measurements

  • Assess correlation with known downstream targets' activation

Recommended approach: When results conflict, design experiments that combine methods—such as cell fractionation followed by Western blot and parallel immunofluorescence—to resolve spatial and quantitative discrepancies .

What experimental approaches can detect interaction partners of phosphorylated ABL1-Y245 in different cellular compartments?

Investigating interaction partners requires specialized techniques:

• Co-immunoprecipitation strategies:

  • Use phospho-ABL1 (Y245) antibody for IP followed by mass spectrometry

  • Perform reverse IPs with suspected binding partners

  • Include appropriate controls (IgG, non-phosphorylatable mutants)

  • Validate with proximity ligation assays for in situ confirmation

• Subcellular fractionation approaches:

  • Separate nuclear, cytoplasmic, mitochondrial, and cytoskeletal fractions

  • Perform IPs from each fraction separately

  • Use biochemical verification of fraction purity

  • Phospho-ABL1 is known to localize to multiple compartments including cytoplasm, nucleus, nuclear membrane, mitochondria, and cytoskeleton

• Phospho-proteomics integration:

  • Compare phosphorylation patterns in interacting proteins

  • Use quantitative phospho-proteomics to identify potential substrates

  • Filter phosphopeptides to focus on tyrosine phosphorylation events (typically ~1.8% of total phosphorylation events)

  • Integrate with protein-protein interaction databases

• Functional validation:

  • Use kinase inhibitors to confirm dependence of interactions on ABL1 activity

  • Create phosphomimetic (Y245D/E) and non-phosphorylatable (Y245F) mutants

  • Compare interaction profiles between wild-type and mutant ABL1

Method highlight: Confocal microscopy with spectral unmixing can effectively distinguish between phospho-ABL1 speckle patterns and determine colocalization with potential binding partners across different cellular compartments .

What sample preparation protocols maximize phospho-ABL1 (Y245) detection in cell lysates?

Effective sample preparation is crucial for preserving phosphorylation status:

• Cell lysis protocol:

  • Harvest cells rapidly to minimize phosphatase activity

  • Use ice-cold lysis buffer containing:

    • Strong phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

    • Protease inhibitor cocktail

    • 1% NP-40 or similar non-denaturing detergent

  • Maintain consistent cell density (4 × 10^7 cells/ml recommended for some assays)

  • Process immediately on ice with minimal handling time

• Buffer composition optimization:

  • Include stabilizing agents: 50% glycerol and 0.5% BSA help maintain antibody stability

  • For storage, include 0.02% sodium azide as a preservative

  • Confirm buffer compatibility with downstream applications

• Protein quantification and normalization:

  • Use methods resistant to phosphatase inhibitor interference

  • Prepare aliquots to avoid freeze-thaw cycles

  • Standardize loading based on total protein rather than single housekeeping proteins

  • Consider using stain-free gel technology for normalization

Validation approach: Compare fresh lysates to those frozen for different periods to establish acceptable storage parameters for your specific cell system.

How can I optimize immunofluorescence protocols to detect low-abundance phospho-ABL1 (Y245) signals?

Low-abundance phosphorylation detection requires enhanced sensitivity:

• Fixation optimization:

  • Compare paraformaldehyde (4%) vs. methanol fixation

  • Test dual fixation methods if needed

  • Minimize fixation time to prevent epitope masking

• Permeabilization considerations:

  • Test different detergents (Triton X-100, Tween-20, saponin)

  • Optimize concentration and exposure time

  • Consider antigen retrieval methods if using paraformaldehyde

• Signal amplification strategies:

  • Tyramide signal amplification for enzymatic enhancement

  • Longer primary antibody incubation (overnight at 4°C)

  • Test different antibody concentrations (1:10-50 for IF/ICC applications)

  • Use high-sensitivity detection systems (e.g., Alexa Fluor 488-conjugated secondary antibodies)

• Imaging parameters:

  • Use confocal microscopy with appropriate filters

  • Optimize detector gain settings

  • Implement deconvolution algorithms

  • Consider photobleaching minimization strategies

Advanced approach: For cells with extremely low signal, implement spinning disk confocal microscopy with EM-CCD detection or consider super-resolution techniques like STED or STORM for improved signal-to-noise ratio.

What considerations are important when designing phospho-ABL1 (Y245) experiments in primary tissue samples?

Working with primary tissues presents unique challenges:

• Tissue preservation protocol:

  • Rapid fixation is critical (within minutes of collection)

  • Flash-freezing samples for biochemical analysis

  • Record and standardize ischemia time

  • Consider phosphatase activity in different tissue types

• Antigen retrieval optimization:

  • Compare heat-induced vs. enzymatic retrieval methods

  • Buffer pH significantly impacts phosphoepitope detection

  • Test microwave, pressure cooker, and water bath methods

  • Validate with positive control tissues (e.g., cancers known to have ABL1 activation)

• Background reduction strategies:

  • Implement endogenous peroxidase quenching for IHC-P

  • Use biotin-blocking systems with avidin-biotin detection

  • Consider mouse-on-mouse blocking for mouse tissues

  • Test different dilutions (1:50-100 recommended for IHC-P)

• Controls and validation:

  • Adjacent sections with phosphatase treatment

  • Known positive tissue controls

  • Peptide competition controls

  • Parallel Western blot validation when possible

Method comparison: For human cancer tissue, formalin-fixed paraffin-embedded samples can be effectively analyzed using peroxidase-conjugated secondary antibodies followed by AEC staining, as demonstrated with breast carcinoma and hepatocarcinoma tissues .