Phospho-MARCKS (S163) Antibody

What is MARCKS and what cellular functions does it regulate?

MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate) is a major PKC substrate expressed in many cell types, with an approximate molecular weight of 32 kDa (can appear as 75-87 kDa on gels depending on cell type). It functions as a plasma membrane-bound protein that dissociates upon phosphorylation by various PKC isoforms. MARCKS plays critical roles in cell motility, cell adhesion, phagocytosis, membrane traffic, and mitogenesis . It serves as a filamentous (F) actin crosslinking protein and has been found to bind calmodulin, actin, and synapsin . In specific systems like neurulation, MARCKS maintains neuroepithelial polarity through stabilization of subapical F-actin .

What is the significance of the Ser163 phosphorylation site on MARCKS?

Serine 163 is one of several phosphorylation sites on MARCKS that can be modified by PKC in response to growth factors and oxidative stress. In human MARCKS, PKC phosphorylates Ser159, 163, 167, and 170 . The phosphorylation at these sites regulates the calcium/calmodulin binding and filamentous (F)-actin cross-linking activities of MARCKS. Importantly, phosphorylation of these sites by PKC results in translocation of MARCKS from the plasma membrane to the cytoplasm , a key regulatory mechanism that affects its function in signal transduction pathways.

How specific is the Phospho-MARCKS (S163) antibody?

The Phospho-MARCKS (S163) antibody is designed to detect MARCKS only when phosphorylated at the Ser163 position. According to product information, these antibodies are typically affinity-purified from rabbit antiserum using epitope-specific immunogens, with purity >95% by SDS-PAGE . The specificity is demonstrated through various validation methods including Western blot analysis of cell lysates treated with PKC activators like PMA, which increases phosphorylation at this site .

What are the recommended applications and dilutions for Phospho-MARCKS (S163) antibody?

Based on product information, the Phospho-MARCKS (S163) antibody can be used for the following applications with the recommended dilutions:

• Western Blotting (WB): 1:500-1:1000

• Immunohistochemistry (IHC): 1:50-1:200

• ELISA: 1:10000

The antibody typically comes in PBS with 0.1% Sodium Azide and 50% Glycerol, and should be stored at 4°C for short term use or aliquoted and stored at -20°C for long term storage, avoiding freeze-thaw cycles .

What controls should be included when validating Phospho-MARCKS (S163) antibody specificity?

For rigorous validation of phospho-specific antibodies like Phospho-MARCKS (S163), the following controls are recommended:

• Positive Controls: Cell lysates treated with PKC activators such as PMA (phorbol 12-myristate 13-acetate), which increases MARCKS phosphorylation at Ser163 .

• Negative Controls:

  • PKC knockout cell lines or PKC inhibitor-treated cells

  • Untreated cell lysates showing baseline phosphorylation levels

• Peptide Competition Assays: Preincubation of the antibody with phosphopeptide versus non-phosphopeptide :

  • The phosphopeptide should abolish immunoreactivity

  • The otherwise identical dephosphopeptide should not affect detection

• Enzymatic Dephosphorylation Controls: Treatment of samples with alkaline phosphatase to remove phosphate groups should eliminate detection by phospho-specific antibodies .

• Genetic Controls: When possible, MARCKS knockout cell lines can demonstrate absolute specificity .

How can I effectively use the Phospho-MARCKS (S163) antibody in Western blotting?

For optimal Western blotting results with Phospho-MARCKS (S163) antibody:

• Sample Preparation:

  • For positive controls, treat cells with PMA (a PKC activator)

  • Multiple cell types have been validated including MCF-7, sp2/0, and PC12 cells

  • Use appropriate lysis buffers that preserve phosphorylation status with phosphatase inhibitors

• SDS-PAGE Conditions:

  • Be aware that MARCKS can appear at different molecular weights: ~32 kDa (theoretical), but often at 75-87 kDa in human samples, and approximately 80 kDa in human and 75 kDa in mouse/rat samples

  • Use appropriate percentage gels that resolve these molecular weight ranges

• Transfer and Detection:

  • Follow standard transfer protocols for proteins in this molecular weight range

  • Use the recommended dilution (1:500-1:1000) in appropriate blocking buffer

  • Include positive controls (PMA-treated cells) alongside experimental samples

• Verification:

  • Consider parallel blotting with total MARCKS antibody to normalize phospho-signal

  • When evaluating signaling pathways, examine both rapid (minutes) and sustained (hours) phosphorylation kinetics

How can I use Phospho-MARCKS (S163) antibody to study PKC signaling pathways?

To effectively study PKC signaling using Phospho-MARCKS (S163) antibody:

• Temporal Dynamics Analysis:

  • MARCKS phosphorylation can be used as a readout for PKC activation

  • Design time-course experiments (3, 5, 10, and 30 minutes) to catch early phosphorylation events in signaling pathways

  • Consider both rapid and sustained phosphorylation kinetics

• PKC Isoform Specificity:

  • In NIH/3T3 fibroblasts, PKC α and PKC ε (but not PKC δ) are responsible for MARCKS phosphorylation

  • Use selective PKC isoform inhibitors or knockout models to determine which isoforms phosphorylate MARCKS at S163 in your system

• Pathway Integration:

  • Monitor correlations with other phosphorylation events

  • MARCKS phosphorylation can be linked to Akt signaling pathways

  • Analyze relationships between MARCKS phosphorylation and cellular functions such as ROS production in monocytic cells

• Optogenetic Approaches:

  • Recent advances include optogenetic control of PKC isoforms (like Opto-PKCε)

  • Studies have shown that Opto-PKCε substrates overlap with endogenous PKCε substrates including MARCKS

What high-throughput methods can incorporate Phospho-MARCKS (S163) antibody for signaling studies?

Several high-throughput approaches have been validated for phospho-specific antibodies including those for MARCKS:

• Reverse Phase Protein Array (RPMA):

  • Can be used to measure phosphorylation levels of multiple proteins simultaneously

  • Has been validated for MARCKS phosphorylation studies in patient samples

  • Allows for comparison of phosphorylation patterns across experimental conditions

• High-Throughput Microscopy (HTM) with Machine Learning:

  • Cells can be seeded in 96-well plates, immunostained, and images acquired using automated microscopy

  • Analysis can be performed using software like CellProfiler to quantify phosphorylation signals

  • This method provides an unbiased, reproducible approach for antibody validation

• Phosphoproteomics:

  • MARCKS phosphorylation at S163 has been detected in large-scale phosphoproteomic studies

  • Table of phosphoproteins including MARCKS S163 shows differential phosphorylation in response to various TLR ligands :

How can I use Phospho-MARCKS (S163) antibody to investigate neurological disorders or cancer?

MARCKS plays important roles in various pathological conditions that can be investigated using phospho-specific antibodies:

• Neurological Studies:

  • MARCKS is necessary for gastrulation and neurulation morphogenetic movements in mice, frogs, and other models

  • Phosphorylation by PKC strongly impairs cell polarity in neuroepithelium

  • The overexpression of nonphosphorylatable MARCKS can revert cellular defects observed after PKC activation

  • Design experiments comparing phosphorylation levels in normal versus pathological tissues

• Cancer Research:

  • Altered PKC signaling is implicated in various cancers

  • Studying MARCKS phosphorylation can reveal dysregulated signaling pathways

  • Use patient-derived samples to compare phosphorylation patterns between normal and cancer tissues

  • Combine with other markers to develop phospho-protein signatures for cancer subtypes

• Inflammation and Immune Response:

  • MARCKS plays a crucial role in monocytic ROS production

  • Long-term TNF pre-incubation enhances monocytic ROS production, which can be blocked in MARCKS and PKCβ knockout cells

  • Design experiments to investigate how MARCKS phosphorylation changes during inflammatory responses

Why might I observe multiple bands or unexpected molecular weights when using Phospho-MARCKS (S163) antibody?

Multiple bands or unexpected molecular weights with Phospho-MARCKS (S163) antibody could result from:

How can I optimize immunostaining protocols for Phospho-MARCKS (S163) antibody?

For optimal immunostaining results:

• Fixation and Permeabilization:

  • For membrane-associated proteins like MARCKS, test different fixation methods

  • PFA/methanol fixation with saponin permeabilization has been shown to work well for membrane-associated synaptic proteins

• Antigen Retrieval:

  • For FFPE tissues, appropriate antigen retrieval methods may be necessary

  • Test both heat-induced epitope retrieval and enzymatic methods

• Blocking Conditions:

  • Use 0.5% BSA in appropriate buffers as per product recommendations

  • Ensure adequate blocking to reduce background

• Antibody Dilution and Incubation:

  • Start with recommended dilutions (1:50-1:200 for IHC)

  • Optimize incubation time and temperature

  • Consider overnight incubation at 4°C for better signal-to-noise ratio

• Detection Systems:

  • Choose secondary antibodies with appropriate conjugates

  • For fluorescence microscopy, select fluorophores with minimal spectral overlap if performing multiplex staining

How should I interpret phospho-MARCKS results in the context of PKC signaling studies?

When interpreting results:

• Baseline Considerations:

  • Establish baseline phosphorylation levels in your cell system

  • Different cell types may have varying levels of basal PKC activity

• Stimulation-Dependent Changes:

  • Phosphorylation typically increases after PKC activation (e.g., by PMA)

  • The timing of phosphorylation can vary (rapid versus sustained)

  • Consider the dynamics of both phosphorylation and dephosphorylation

• Functional Correlation:

  • Correlate phosphorylation with membrane translocation of MARCKS

  • MARCKS phosphorylation leads to its dissociation from the plasma membrane

  • This translocation affects its interactions with F-actin and other binding partners

• Pathway Context:

  • MARCKS phosphorylation should be interpreted in the context of other signaling events

  • In TNF-stimulated monocytes, MARCKS phosphorylation correlates with enhanced ROS production

  • In neurulation, MARCKS phosphorylation is associated with impaired cell polarity

• Quantification Approaches:

  • Normalize phospho-MARCKS signal to total MARCKS when possible

  • For Western blots, use densitometry with appropriate controls

  • For microscopy, employ quantitative image analysis as described in high-throughput validation studies