MARCKS Antibody

What is MARCKS protein and why is it an important research target?

MARCKS is a membrane-associated protein that plays critical roles in structural modulation of the actin cytoskeleton, chemotaxis, motility, cell adhesion, phagocytosis, and exocytosis through lipid sequestering and protein docking mechanisms. Its influence extends to embryonic development, tissue regeneration, neuronal plasticity, and inflammation. MARCKS sequesters phosphatidylinositol 4,5-bisphosphate (PIP2) at lipid rafts in plasma membranes, which is reversed by protein kinase C (PKC) phosphorylation. During inflammation, it promotes migration and adhesion of inflammatory cells and secretion of cytokines, particularly in macrophages. MARCKS also participates in neurite initiation and outgrowth regulation through interaction with CDC42 and other components that modulate cytoskeletal structures .

Which applications are most suitable for MARCKS antibodies?

MARCKS antibodies are widely used in multiple applications:

• Western Blot (WB): Typically used at dilutions around 1:10000, detecting the ~80-83 kDa MARCKS protein

• Immunohistochemistry (IHC): Used at dilutions between 1:500-1:1000

• Immunocytochemistry/Immunofluorescence (ICC/IF): Effective at dilutions ranging from 1:500-1:5000

• Immunoprecipitation (IP): For isolating MARCKS protein complexes

• ELISA: For quantitative measurement of MARCKS protein levels

The choice of application should be guided by specific research questions and sample types.

How do I select the appropriate MARCKS antibody for my specific research?

When selecting a MARCKS antibody, consider:

• Host Species: Chicken and rabbit polyclonal antibodies are commonly used

• Reactivity: Confirm compatibility with your species of interest (human, mouse, rat, etc.)

• Target Region: Choose between total MARCKS or phospho-specific antibodies depending on your research focus

• Application Validation: Verify the antibody has been validated for your intended application

• Immunogen: Consider whether the antibody was raised against full-length protein (better for detecting total MARCKS) or specific peptide sequences (which may provide higher specificity for certain domains)

For phosphorylation studies, use antibodies specific to particular phosphorylation sites, such as S152/S156 (equivalent to S167/S170 in some numbering systems) .

What is the optimal protocol for Western blot detection of MARCKS?

For optimal Western blot detection of MARCKS:

• Sample Preparation:

  • Extract proteins using standard lysis buffers containing protease inhibitors

  • For phospho-MARCKS detection, add phosphatase inhibitors to prevent dephosphorylation

• Gel Electrophoresis:

  • Use 10% SDS-PAGE gels (MARCKS migrates at ~80-83 kDa)

  • Load 20-50 μg of total protein per well

• Transfer and Blocking:

  • Transfer to PVDF or nitrocellulose membranes

  • Block in 5% milk/TBS (for total MARCKS) or 5% BSA/TBS (for phospho-MARCKS)

• Antibody Incubation:

  • Primary antibody: Use at 1:10000 dilution in 5% milk/TBS or 5% BSA/TBS

  • Secondary antibody: HRP-conjugated at 1:5000-1:10000 dilution

• Detection:

  • Use enhanced chemiluminescence (ECL) detection system

  • Phospho-specificity can be confirmed by treating samples with lambda phosphatase as a negative control

How can I optimize immunohistochemistry protocols for MARCKS detection?

For optimal IHC detection of MARCKS:

• Tissue Preparation:

  • Fix tissues in 4% paraformaldehyde or 10% neutral buffered formalin

  • Paraffin embedding is suitable for most applications

• Antigen Retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Boil for 15-20 minutes followed by cooling to room temperature

• Blocking and Antibody Incubation:

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Block nonspecific binding with 5-10% normal serum

  • Incubate with primary antibody at 1:500-1:1000 dilution overnight at 4°C

  • Use appropriate HRP-conjugated secondary antibody

• Detection and Counterstaining:

  • Develop signal using DAB substrate

  • Counterstain with hematoxylin

  • Mount with appropriate mounting medium

What controls should be included when using MARCKS antibodies?

Essential controls for MARCKS antibody experiments include:

• Positive Controls:

  • Brain tissue lysates (MARCKS is highly expressed in neurons)

  • Cell lines known to express MARCKS (e.g., macrophages)

• Negative Controls:

  • MARCKS knockout cells (CRISPR/Cas9-generated ΔMARCKS cell lines)

  • Primary antibody omission

  • IgG isotype control

• Phosphorylation-Specific Controls:

  • Treatment with lambda phosphatase to remove phosphate groups

  • Stimulation with PKC activators (e.g., PMA) to increase phosphorylation

  • Inhibitors of PKC to decrease phosphorylation

• Validation Controls:

  • Peptide competition assays to confirm antibody specificity

  • Multiple antibodies targeting different epitopes to confirm results

How can MARCKS antibodies be used to study inflammation processes?

MARCKS plays a key role in inflammation, and antibodies can be used to investigate:

• Cytokine Production Analysis:

  • Compare cytokine levels (TNF, IL-6) in wild-type versus MARCKS knockout macrophages after LPS stimulation using ELISA

  • Use phospho-MARCKS antibodies to correlate MARCKS phosphorylation state with cytokine production

• Inflammatory Cell Migration Studies:

  • Perform immunofluorescence staining with MARCKS antibodies to visualize redistribution during cell migration

  • Combine with live-cell imaging to track MARCKS dynamics during inflammatory responses

• Signaling Pathway Analysis:

  • Use Western blotting with phospho-MARCKS and other signaling molecules (pAkt, pERK1/2, pBTK, pPLCγ2, pSyk) to map inflammation-related signaling cascades

  • Generate a timeline of phosphorylation events using time-course experiments

• Therapeutic Intervention Assessment:

  • Apply MARCKS-targeted therapeutics and use antibodies to measure changes in MARCKS expression and phosphorylation

  • Correlate with inflammatory markers to assess efficacy

Research has demonstrated that MARCKS knockout macrophages show decreased production of pro-inflammatory cytokines (TNF and IL-6) after LPS stimulation, suggesting MARCKS is a key regulator of inflammation whose inhibition might benefit inflammatory disease treatment .

What methodologies can be employed to study MARCKS phosphorylation states in different cellular contexts?

To study MARCKS phosphorylation states:

• Phospho-specific Antibody Selection:

  • Use antibodies targeting key phosphorylation sites (S152/S156 or S167/S170)

  • These sites are within the effector domain that regulates calmodulin binding

• Stimulation Protocols:

  • PKC activators (PMA, bryostatin)

  • Calcium ionophores

  • Physiological stimuli (growth factors, inflammatory mediators)

  • B-cell receptor (BCR) activation

• Phosphorylation Dynamics Analysis:

  • Time-course Western blots to track phosphorylation kinetics

  • Immunofluorescence to visualize subcellular redistribution after phosphorylation

  • FRET-based biosensors for real-time monitoring in live cells

• Phosphorylation-Function Correlation:

  • Combine phospho-MARCKS detection with functional assays (migration, adhesion, cytokine production)

  • Use phospho-mimetic and phospho-resistant MARCKS mutants as controls

• Mass Spectrometry Validation:

  • Immunoprecipitate MARCKS using total MARCKS antibodies

  • Analyze phosphorylation sites by mass spectrometry to confirm antibody specificity

How can I use MARCKS antibodies in the context of neurodevelopmental studies?

For neurodevelopmental research:

• Neurite Outgrowth Analysis:

  • Culture primary neurons or neuronal cell lines

  • Perform immunofluorescence with MARCKS antibodies to visualize distribution during neurite formation

  • Quantify co-localization with CDC42 and other cytoskeletal regulators

• Axon Development Studies:

  • Use live-cell imaging with fluorescently tagged MARCKS antibodies (if available) to track vesicle docking and fusion

  • Investigate co-localization with RAB10-positive vesicles

  • Analyze the effects of MARCKS knockdown/knockout on axonal development

• Synaptic Plasticity Investigations:

  • Perform immunohistochemistry on brain sections to analyze MARCKS expression at different developmental stages

  • Use electron microscopy with immunogold-labeled MARCKS antibodies to study synaptic localization

• Activity-Dependent Phosphorylation:

  • Stimulate neurons with glutamate or KCl to induce activity

  • Analyze changes in MARCKS phosphorylation using phospho-specific antibodies

  • Correlate with functional changes in synaptic transmission

What are common issues with MARCKS antibody specificity and how can they be addressed?

Common specificity issues and solutions:

• Multiple Bands in Western Blot:

  • Cause: MARCKS can undergo post-translational modifications or degradation

  • Solution: Use freshly prepared samples with protease inhibitors; verify specificity with MARCKS knockout controls; optimize primary antibody concentration

• Cross-Reactivity:

  • Cause: Antibody binds to similar epitopes in other proteins

  • Solution: Perform peptide competition assays; use multiple antibodies targeting different MARCKS epitopes; validate with siRNA knockdown

• Variable Results Across Different Tissues:

  • Cause: Tissue-specific post-translational modifications or MARCKS isoforms

  • Solution: Use tissue-specific positive controls; optimize extraction and fixation protocols for each tissue type

• Phospho-Specificity Issues:

  • Cause: Incomplete phosphorylation or phosphatase activity during sample preparation

  • Solution: Include phosphatase inhibitors; verify phospho-specificity using lambda phosphatase treatment as demonstrated in Western blots; use appropriate stimuli to induce phosphorylation as positive controls

How do I interpret conflicting results when using different MARCKS antibodies?

When facing conflicting results:

• Epitope Mapping Analysis:

  • Determine the exact epitopes recognized by each antibody

  • Different antibodies may recognize distinct domains or conformations of MARCKS

• Post-Translational Modification Consideration:

  • Check if antibodies are sensitive to phosphorylation, myristoylation, or other modifications

  • Some antibodies may have reduced binding when MARCKS is phosphorylated

• Validation Strategy:

  • Use MARCKS knockout/knockdown systems as definitive controls

  • Employ multiple techniques (WB, IHC, IF) to cross-validate findings

  • Consider orthogonal approaches (mRNA expression, mass spectrometry)

• Application-Specific Optimization:

  • Different antibodies may perform optimally in specific applications

  • Test multiple fixation and extraction methods to determine if conflicts are methodology-dependent

• Batch Variation Assessment:

  • Compare lot numbers and manufacturing dates

  • Request validation data from manufacturers for specific lots

What statistical considerations should be applied when quantifying MARCKS expression or phosphorylation?

For robust statistical analysis:

How can MARCKS antibodies be employed in antibody-antigen complex modeling studies?

For antibody-antigen complex modeling:

• Structural Data Collection:

  • Use MARCKS antibodies in crystallography studies to determine antibody-antigen complex structures

  • Apply cryo-electron microscopy for larger complexes

• ML-Driven Modeling Approaches:

  • Employ machine learning tools like AlphaFold2-Multimer, ABodyBuilder2, or IgFold to predict antibody-antigen interactions

  • Generate ensembles of models to account for structural flexibility

• Docking Protocol Implementation:

  • Use information-driven protocols like HADDOCK with data on paratope and epitope residues

  • Employ rigid-body docking followed by flexible refinement for improved accuracy

• Ensemble Method Advantages:

  • Utilize multiple antibody models in docking to improve success rates

  • Consider clustering different antibody structures (CLE protocol) for enhanced performance

• Validation Approaches:

  • Calculate interface, paratope, and epitope RMSD relative to experimental structures

  • Use confidence scores from modeling tools to predict model quality and docking success

Current research demonstrates ensemble approaches significantly improve docking performance in antibody-antigen complex modeling.

What are the latest methodologies for using MARCKS antibodies in proteomic analyses?

Advanced proteomic approaches include:

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Use MARCKS antibodies to immunoprecipitate protein complexes

  • Identify binding partners through mass spectrometry

  • Compare interactomes under different conditions (e.g., phosphorylated vs. non-phosphorylated)

• Proximity-Dependent Labeling:

  • Generate MARCKS-BioID or MARCKS-APEX2 fusion proteins

  • Use antibodies to validate proximity labeling results

  • Map the MARCKS proximal proteome in different cellular compartments

• Cross-Linking Mass Spectrometry (XL-MS):

  • Cross-link MARCKS with its binding partners in situ

  • Immunoprecipitate with MARCKS antibodies

  • Identify cross-linked peptides by mass spectrometry to map interaction interfaces

• Phosphoproteomics Integration:

  • Combine MARCKS immunoprecipitation with phosphopeptide enrichment

  • Analyze phosphorylation dynamics in MARCKS and associated proteins

  • Correlate with functional outcomes in different cellular contexts

Recent proteomic studies comparing wild-type and MARCKS knockout macrophages revealed MARCKS involvement in specific biological processes including innate immune response, inflammatory response, cytokine production, and molecular functions such as ATP-gated cation channel activity and oxidoreductase activity.

How can I develop multi-parameter imaging protocols using MARCKS antibodies?

For advanced imaging protocols:

• Multiplex Immunofluorescence Development:

  • Combine MARCKS antibodies with markers for specific cellular compartments or signaling molecules

  • Use spectral unmixing to resolve overlapping fluorophores

  • Establish sequential staining protocols if antibodies are from the same species

• Super-Resolution Microscopy Application:

  • Use MARCKS antibodies with super-resolution techniques (STORM, PALM, SIM)

  • Visualize nanoscale distribution of MARCKS at the plasma membrane

  • Co-localize with PIP2 and other binding partners at high resolution

• Live-Cell Imaging Optimization:

  • Use fluorescently-labeled MARCKS antibody fragments for live-cell applications

  • Track MARCKS redistribution in response to stimuli in real-time

  • Combine with optogenetic tools to manipulate MARCKS function

• Correlative Light and Electron Microscopy (CLEM):

  • Localize MARCKS using immunofluorescence

  • Process the same sample for electron microscopy

  • Correlate MARCKS distribution with ultrastructural features

• Image Analysis Workflows:

  • Develop automated segmentation algorithms for MARCKS-positive structures

  • Quantify co-localization with binding partners

  • Track changes in MARCKS distribution over time using consistent metrics

How are MARCKS antibodies being used to investigate the role of MARCKS in disease models?

MARCKS antibodies are being applied to study disease associations through:

• Cancer Research Applications:

  • Compare MARCKS expression and phosphorylation across tumor types

  • Correlate with tumor progression, invasion, and metastasis

  • Evaluate as a potential biomarker for specific cancer subtypes

• Neurodegenerative Disease Models:

  • Analyze MARCKS expression in Alzheimer's and Parkinson's disease models

  • Investigate interactions with disease-associated proteins

  • Study the role in neuroinflammatory processes

• Inflammatory Disease Research:

  • Examine MARCKS as a therapeutic target in inflammatory conditions

  • Use antibodies to monitor intervention efficacy

  • Study MARCKS-related signaling in autoimmune disorders

• Hematological Malignancy Studies:

  • Investigate MARCKS in chronic lymphocytic leukemia (CLL) response to BTK inhibitors

  • Analyze how MARCKS affects cell motility and BCR signaling in CLL

  • Use phospho-specific antibodies to monitor treatment response

Recent findings suggest MARCKS acts as a fine-tuning element of the B-cell receptor via PIP2 interaction, and its expression levels in CLL may predict response to therapies like acalabrutinib.

What are the methodological considerations for validating MARCKS knockout models using antibodies?

For validating knockout models:

• Multiple Validation Approach:

  • Use Western blotting with antibodies targeting different MARCKS epitopes

  • Perform immunohistochemistry/immunofluorescence on knockout tissues

  • Conduct mass spectrometry analysis to confirm complete protein absence

• Off-Target Effect Assessment:

  • Check for compensatory upregulation of MARCKS-like proteins

  • Evaluate changes in related signaling pathways

  • Use multiple knockout strategies (CRISPR, shRNA, siRNA) and compare results

• Rescue Experiment Design:

  • Re-express MARCKS in knockout cells to confirm phenotype reversal

  • Use antibodies to verify expression levels in rescue experiments

  • Include phosphorylation-site mutants to investigate specific functions

• Knockout Verification Methods:

  • Genomic PCR to confirm targeted modification

  • mRNA expression analysis through RT-PCR or RNA-seq

  • Protein absence confirmation through Western blot with multiple antibodies

  • Functional assays demonstrating loss of MARCKS-dependent functions

Recent studies successfully generated MARCKS knockout in immortalized macrophages (IMMs) using CRISPR-Cas9, confirming knockout through Western blot and mass spectrometry, and demonstrated functional consequences through cytokine response assays.

How can MARCKS antibodies be combined with genetic approaches for comprehensive functional studies?

Integrative approaches include:

• Antibody-Validated CRISPR Screens:

  • Perform CRISPR screens targeting MARCKS interactors or regulators

  • Use antibodies to validate hits and assess effects on MARCKS expression/phosphorylation

  • Combine with functional assays to establish mechanism

• Conditional Knockout Validation:

  • Generate tissue-specific or inducible MARCKS knockout models

  • Use antibodies to confirm knockout efficiency in specific tissues/timepoints

  • Correlate with phenotypic changes

• Structure-Function Analysis:

  • Express MARCKS mutants (phosphorylation sites, PIP2 binding, myristoylation)

  • Use antibodies to assess expression levels and subcellular localization

  • Evaluate functional consequences of specific domain alterations

• Signaling Pathway Integration:

  • Combine genetic manipulation of upstream regulators/downstream effectors

  • Use phospho-specific antibodies to map signaling networks

  • Create comprehensive pathway models based on antibody-validated interactions

What approaches can be used to compare different types of MARCKS antibodies for specific research applications?

For comprehensive antibody comparison:

• Cross-Validation Protocol:

  • Test multiple antibodies (different hosts, clonality, epitopes) on the same samples

  • Compare staining patterns, signal intensity, and background

  • Use knockout controls to establish specificity baseline

• Systematic Comparison Workflow:

  • Create a standardized panel of positive and negative controls

  • Test each antibody across multiple applications (WB, IHC, ICC, IP)

  • Generate quantitative performance metrics for objective comparison

• Application-Specific Optimization:

  • For each application, determine optimal conditions for each antibody

  • Consider fixation methods, blocking agents, incubation times, and detection systems

  • Document optimal protocols for future reference

How should researchers interpret MARCKS antibody data in the context of other experimental evidence?

For comprehensive data interpretation:

• Multi-Level Validation Approach:

  • Confirm antibody-based findings with orthogonal techniques

  • Correlate protein-level data with mRNA expression

  • Use genetic manipulation to establish causality

• Functional Correlation Framework:

  • Link MARCKS expression/phosphorylation changes to functional outcomes

  • Consider context-dependent functions in different cell types

  • Account for temporal dynamics in signaling responses

• Integrated Data Analysis:

  • Combine antibody-based data with genomic, transcriptomic, and proteomic datasets

  • Use systems biology approaches to place MARCKS in broader networks

  • Apply computational modeling to predict functional consequences

• Contradictory Results Resolution:

  • Systematically evaluate potential sources of discrepancy

  • Consider differences in experimental conditions, cell types, or stimuli

  • Use multiple antibodies and techniques to resolve conflicts

  • Distinguish between correlation and causation in interpreting results