Phospho-LIMK2 (Thr505) Antibody

What is LIMK2 and what biological significance does its phosphorylation at Thr505 indicate?

LIMK2 (LIM domain kinase 2) is a serine/threonine kinase that plays a critical role in cytoskeletal dynamics, particularly in regulating actin filament stability. LIMK2 belongs to a family of proteins containing LIM motifs, which are typically involved in cell fate determination and growth control .

Phosphorylation at Threonine 505 represents an activation marker for LIMK2, indicating that the kinase has been switched to its catalytically active state. When phosphorylated at Thr505, LIMK2 can phosphorylate downstream targets, most notably cofilin, which regulates the stabilization of F-actin structures . This phosphorylation event is therefore a key indicator of active cytoskeletal reorganization processes within the cell, including those involved in cell motility, morphogenesis, and various cellular responses to external stimuli .

What are the primary applications for Phospho-LIMK2 (Thr505) Antibody in research settings?

Phospho-LIMK2 (Thr505) Antibody serves multiple experimental applications in research:

• Western Blotting: Commonly used at dilutions between 1:500-1:1000 to detect activated LIMK2 in cell or tissue lysates . This technique allows quantification of phosphorylation levels under different experimental conditions.

• Immunohistochemistry (IHC): Applied at dilutions of 1:50-1:100 to visualize the tissue and cellular distribution of phosphorylated LIMK2 .

• Immunofluorescence (IF): Enables subcellular localization studies of activated LIMK2, particularly in relation to cytoskeletal structures .

• ELISA: Used at higher dilutions (approximately 1:10000) for quantitative analysis of phosphorylated LIMK2 levels .

• Immunocytochemistry: Allows for detailed cellular localization studies of activated LIMK2 .

How should Phospho-LIMK2 (Thr505) Antibody be stored and handled to maintain optimal activity?

For optimal preservation of antibody activity:

• Store at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles which can reduce antibody efficacy

• When working with the antibody, keep it on ice or at 4°C

• Most formulations are supplied in PBS with stabilizers, and this buffer composition should be maintained

• Follow manufacturer's guidelines for specific lot information

• For western blotting applications, prepare working dilutions fresh before use

• Monitor expiration dates provided by manufacturers

What are the recommended protocols for using Phospho-LIMK2 (Thr505) Antibody in Western blot applications?

Western Blot Protocol for Phospho-LIMK2 (Thr505) Detection:

• Sample Preparation:

  • Cells should be lysed in buffer containing phosphatase inhibitors to preserve phosphorylation status

  • Include positive controls (e.g., PMA-treated HeLa cells)

  • Load 20-40 μg of total protein per lane

• Gel Electrophoresis and Transfer:

  • Use 8-10% SDS-PAGE gels for optimal resolution around 70-72 kDa

  • Transfer to PVDF or nitrocellulose membranes using standard protocols

• Antibody Incubation:

  • Block membrane with 5% BSA in TBST (not milk, which contains phosphatases)

  • Dilute primary antibody 1:500-1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3-5 times with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody (typically anti-rabbit)

• Detection:

  • Develop using ECL substrate

  • Expected molecular weight: approximately 70-72 kDa

• Validation Controls:

  • Run parallel blots with total LIMK2 antibody to normalize phospho-signals

  • Include phosphatase-treated samples as negative controls

How can researchers effectively design experiments to study LIMK2 phosphorylation dynamics?

Experimental Design Strategies:

• Temporal Analysis:

  • Design time-course experiments (5, 15, 30, 60 min) following stimulation

  • Use stimulus-specific positive controls (e.g., PMA treatment for PKC-mediated pathways)

  • Include both early (seconds to minutes) and late (hours) time points to capture full phosphorylation dynamics

• Pathway Dissection:

  • Employ specific inhibitors to target upstream kinases:

    • ROCK inhibitors (Y-27632)

    • PAK inhibitors

    • Staurosporine (broad kinase inhibitor)

  • Use genetic approaches (siRNA, CRISPR) to knock down pathway components

• Subcellular Localization:

  • Combine phospho-LIMK2 detection with subcellular fractionation

  • Use dual immunofluorescence to co-localize phospho-LIMK2 with cytoskeletal markers or upstream activators

• Functional Correlation:

  • Pair phosphorylation analysis with functional readouts:

    • Actin polymerization assays

    • Cell migration assays

    • Morphological analyses

• Quantification Methods:

  • Employ densitometry for western blots

  • Use high-content imaging for immunofluorescence quantification

  • Consider phospho-flow cytometry for single-cell analysis

What are common challenges in Phospho-LIMK2 (Thr505) Antibody experiments and how can they be resolved?

Challenge 1: Weak or No Signal

• Possible Causes:

  • Insufficient phosphorylated protein

  • Degradation due to phosphatase activity

  • Suboptimal antibody concentration

• Solutions:

  • Use positive controls like PMA-treated cells

  • Ensure phosphatase inhibitors are fresh and active

  • Optimize antibody concentration (try 1:250 for weak signals)

  • Extend primary antibody incubation time

Challenge 2: Non-specific Bands

• Possible Causes:

  • Cross-reactivity with LIMK1 (high homology)

  • Insufficient blocking

  • Degradation products

• Solutions:

  • Extend blocking time using 5% BSA in TBST

  • Perform antibody validation with LIMK2 knockdown samples

  • Run parallel blots with total LIMK2 antibody to confirm band identity

  • Optimize wash steps (increase number and duration)

Challenge 3: Inconsistent Phosphorylation Levels

• Possible Causes:

  • Rapid dephosphorylation during sample preparation

  • Cell density variations

  • Serum components affecting baseline phosphorylation

• Solutions:

  • Standardize cell culture conditions

  • Ensure rapid sample processing with pre-chilled buffers

  • Consider using phosphatase inhibitor cocktails with multiple inhibitors

  • Normalize to total protein and total LIMK2 levels

Challenge 4: Difficulty Detecting Endogenous Levels

• Possible Causes:

  • Low abundance of phosphorylated form

  • Cell type-specific expression patterns

• Solutions:

  • Use enrichment strategies (immunoprecipitation before western blot)

  • Consider more sensitive detection methods (chemiluminescent substrates)

  • Load more total protein (up to 50-60 μg per lane)

How can researchers validate the specificity of Phospho-LIMK2 (Thr505) Antibody?

Validation Approaches:

• Phosphatase Treatment Control:

  • Treat half of your sample with lambda phosphatase

  • Run treated and untreated samples side-by-side

  • The phospho-specific band should disappear in treated samples

• Stimulation/Inhibition Controls:

  • Use ROCK activators to increase LIMK2 phosphorylation

  • Use ROCK inhibitors to decrease phosphorylation

  • Compare band intensities across conditions

• Genetic Validation:

  • Use LIMK2 siRNA or CRISPR knockout models

  • The specific band should be absent in knockout samples

  • Consider LIMK2 T505A mutant transfection (non-phosphorylatable)

• Peptide Competition:

  • Pre-incubate antibody with phospho-peptide containing the Thr505 sequence

  • This should abolish specific binding

• Cross-Reactivity Assessment:

  • Test antibody against samples expressing only LIMK1 to evaluate potential cross-reactivity

  • This is particularly important as some antibodies detect both LIMK1 (Thr508) and LIMK2 (Thr505)

How does LIMK2 phosphorylation at Thr505 differ functionally from LIMK1 phosphorylation at Thr508?

Despite their structural similarities, LIMK1 and LIMK2 exhibit distinct functional characteristics when phosphorylated:

Phosphorylation Comparison:

While both phosphorylated kinases regulate actin dynamics through cofilin phosphorylation, expression patterns suggest they have different functions during development . LIMK1 has been more strongly implicated in neuronal differentiation of PC12 cells, where it appears to interfere with events downstream of MAPK activation . A truncated form of LIMK2 has been identified in adult testis, suggesting tissue-specific functions .

Research using knockout models demonstrates that these kinases are not completely redundant, and their phosphorylation states correlate with distinct cellular processes.

What signaling pathways regulate LIMK2 phosphorylation at Thr505 and how can they be experimentally manipulated?

Key Regulatory Pathways:

• Rho/ROCK Pathway:

  • Primary upstream regulator of LIMK2 phosphorylation

  • Experimental manipulation:

    • RhoA activators (lysophosphatidic acid, calpeptin)

    • ROCK inhibitors (Y-27632, Fasudil)

    • Expression of constitutively active or dominant-negative RhoA constructs

• p21-activated Kinase (PAK) Pathway:

  • Secondary regulator of LIMK2

  • Experimental manipulation:

    • Rac1/Cdc42 activators

    • PAK inhibitors (IPA-3, PF-3758309)

    • Constitutively active PAK1 expression

• Myotonic Dystrophy Kinase-Related CDC42-Binding Kinase (MRCK):

  • Alternative LIMK2 activator

  • Less studied but important in certain contexts

  • Manipulated through CDC42 pathway modulation

• Cross-talk with Other Pathways:

  • MAPKs may indirectly regulate LIMK2 phosphorylation

  • PI3K/Akt pathway can modulate ROCK activity and thus LIMK2 phosphorylation

  • Calcium signaling affects RhoA activation

Experimental Approaches to Study Pathway Regulation:

• Pharmacological:

  • Use pathway-specific inhibitors or activators

  • Design dose-response and time-course experiments

• Genetic:

  • Employ siRNA knockdowns of pathway components

  • Use dominant-negative or constitutively active constructs

  • CRISPR-mediated gene editing of regulatory components

• Physiological Stimuli:

  • Growth factors (PDGF, EGF)

  • Mechanical stimulation (stretch, shear stress)

  • ECM components that activate integrin signaling

How can researchers integrate Phospho-LIMK2 (Thr505) data with cytoskeletal dynamics studies?

Integrated Research Approaches:

• Live-Cell Imaging Combined with Fixed-Cell Phospho-Analysis:

  • Track actin dynamics in living cells using fluorescent reporters

  • Fix cells at key timepoints for phospho-LIMK2 immunostaining

  • Correlate temporal changes in phosphorylation with observed cytoskeletal rearrangements

• Super-Resolution Microscopy Applications:

  • Use techniques like STORM or PALM to co-localize phospho-LIMK2 with cytoskeletal structures

  • Analyze nanoscale spatial relationships between activated LIMK2 and its substrates

• Functional Cytoskeletal Assays:

  • Actin Turnover: Fluorescence recovery after photobleaching (FRAP) of actin structures

  • Migration Analysis: Wound healing or single-cell tracking with phospho-LIMK2 status

  • 3D Matrix Studies: Invasion assays correlated with LIMK2 activation

  • Mechanical Measurements: Atomic force microscopy to correlate cell stiffness with LIMK2 phosphorylation

• Biochemical Activity Correlation:

  • Measure cofilin phosphorylation status (Ser3) as a direct downstream effect

  • Assess F-actin/G-actin ratios in samples with differing phospho-LIMK2 levels

  • Use actin co-sedimentation assays to measure polymerization dynamics

• Multi-Omics Integration:

  • Correlate phosphoproteomics data for LIMK2 with other cytoskeletal regulators

  • Integrate transcriptomics to identify co-regulated networks

  • Use systems biology approaches to model temporal relationships

How should researchers analyze contradictory results when studying LIMK2 phosphorylation patterns?

Systematic Approach to Resolving Contradictions:

• Technical Verification:

  • Confirm antibody specificity with appropriate controls

  • Validate results using alternative detection methods

  • Ensure phosphorylation is preserved during sample preparation

  • Standardize quantification methods across experiments

• Biological Context Evaluation:

  • Cell Type Differences: LIMK2 function may vary between cell types

  • Confluence/Density Effects: Cell density affects actin dynamics and baseline phosphorylation

  • ECM Composition: Different substrates can alter basal phosphorylation states

  • Growth Factor Environment: Serum components influence LIMK2 regulation

• Temporal Considerations:

  • Phosphorylation is dynamic - contradictory results may reflect different time points

  • Design detailed time-course experiments (seconds to hours)

  • Consider oscillatory patterns in signaling pathways

• Pathway Crosstalk Analysis:

  • Map interconnections between LIMK2-regulating pathways

  • Consider compensatory mechanisms activated by experimental perturbations

  • Evaluate feedback loops that may complicate interpretation

• Isoform-Specific Effects:

  • Analyze which LIMK2 isoforms are present in your experimental system

  • Consider that antibodies may have different affinities for various isoforms

  • The truncated testis-specific form may have distinct regulation

What criteria should be used to determine if phospho-LIMK2 levels are physiologically significant?

Evaluation Framework:

• Quantitative Benchmarks:

  • Fold-Change Threshold: Generally, >1.5-2 fold changes are considered biologically meaningful

  • Statistical Significance: Apply appropriate statistical tests with multiple comparisons correction

  • Reproducibility: Consistent results across independent experiments

• Functional Correlation:

  • Changes in phospho-LIMK2 should correlate with:

    • Altered cofilin phosphorylation status

    • Measurable changes in F-actin organization

    • Phenotypic outcomes (e.g., migration rate, morphology changes)

• Dose-Response Relationships:

  • Graded stimuli should produce proportional phosphorylation responses

  • Establish EC50 values for different stimuli

• Temporal Dynamics:

  • Evaluate persistence of phosphorylation (transient vs. sustained)

  • Match kinetics to known biological processes

  • Consider rapid cycling between phosphorylated/dephosphorylated states

• Comparative Analysis:

  • Compare with published literature values

  • Benchmark against known physiological activators

  • Consider relative changes compared to other phosphorylation events in the same pathway

What are the current frontiers in LIMK2 phosphorylation research?

Current research frontiers exploring LIMK2 phosphorylation include:

• Role in Disease Contexts:

  • Cancer invasion and metastasis mechanisms

  • Neurodegenerative disorders involving cytoskeletal dysfunction

  • Cardiac and vascular remodeling pathologies

• Regulatory Mechanisms Beyond Thr505:

  • Additional phosphorylation sites affecting LIMK2 function

  • Interplay between phosphorylation and other post-translational modifications

  • Scaffold proteins that organize LIMK2 signaling complexes

• Tissue-Specific Functions:

  • Specialized roles in neurons vs. epithelial cells vs. immune cells

  • Developmental stage-specific regulation and function

  • Stem cell-specific cytoskeletal regulation

• Subcellular Compartmentalization:

  • Nuclear functions of phosphorylated LIMK2

  • Association with specific actin structures (lamellipodia, filopodia, stress fibers)

  • Role at cell-cell and cell-matrix adhesion sites

• Technological Innovations:

  • Biosensors for real-time LIMK2 activity monitoring

  • Optogenetic approaches to spatiotemporally control LIMK2 activation

  • Single-cell analysis of phosphorylation heterogeneity

How can phospho-LIMK2 research be integrated with broader cytoskeletal and cell biology studies?

Integration Strategies:

• Multi-level Analysis Framework:

  • Link molecular events (LIMK2 phosphorylation) to cellular behaviors

  • Connect cellular behaviors to tissue-level functions

  • Relate tissue functions to organismal physiology

• Interdisciplinary Methodologies:

  • Combine biochemical approaches with biophysical measurements

  • Integrate computational modeling with experimental validation

  • Apply systems biology approaches to position LIMK2 in broader networks

• Technological Integration:

  • Correlative light and electron microscopy to link phospho-signals to ultrastructure

  • Microfluidic systems to control microenvironment while analyzing phosphorylation

  • Organ-on-chip platforms to study LIMK2 in tissue-like contexts

• Translational Applications:

  • Drug discovery targeting LIMK2 phosphorylation

  • Biomarker development based on phospho-LIMK2 status

  • Therapeutic approaches to modulate cytoskeletal dynamics

• Comparative Biology Perspectives:

  • Evolutionary conservation of LIMK2 regulation across species

  • Cell-type specific adaptations of the LIMK2 pathway

  • Specialized cytoskeletal regulation in diverse biological contexts