MARK2 Antibody, HRP conjugated

What is MARK2 and why is it an important research target?

MARK2, also known as EMK1 (ELKL motif kinase 1) or Par1b, belongs to the CAMK Ser/Thr protein kinase family and plays crucial roles in neuronal polarity, cell migration, and cytoskeletal organization . Recent research has identified MARK2 as a master regulator of both actomyosin and microtubule cytoskeletal systems, where it promotes focal adhesion formation to mediate directionally persistent cell motility . MARK2 has also emerged as a candidate diagnostic gene with immune-associated functions . Understanding its multifaceted roles requires specific antibodies that can reliably detect this protein across various experimental platforms.

What are the typical applications for MARK2 antibodies in research?

MARK2 antibodies have been validated for multiple applications including:

This versatility enables researchers to investigate MARK2 expression, localization, and interactions using complementary techniques .

How does HRP conjugation benefit MARK2 antibody applications?

HRP (horseradish peroxidase) conjugation provides several methodological advantages:

These benefits are particularly valuable when examining complex MARK2 interactions with cytoskeletal components and signaling proteins.

What controls should be included when using MARK2 antibodies in experimental systems?

Comprehensive experimental design for MARK2 studies should include:

• Positive controls: Brain tissue lysates from mouse or rat, which express detectable levels of endogenous MARK2

• Negative controls: MARK2-knockout cell lines, which have been validated to show reduced stress fiber formation and significantly lower phosphorylated myosin regulatory light chain (pMRLC) levels

• Loading controls: Standard housekeeping proteins appropriate for the tissue/cell type

• Specificity controls: Pre-adsorption with immunizing peptide or MARK2 siRNA-treated samples, which should show reduced MARK2 signal

When studying MARK2's role in cell migration, researchers should include both wild-type cells and MARK2-KO cells in wound-healing assays to observe differences in stress fiber formation and contractility .

What are the recommended sample preparation methods for MARK2 detection?

For optimal MARK2 detection:

• Tissue extraction: For brain tissue, rapid extraction and flash-freezing maintain protein integrity

• Lysis buffers: Include protease and phosphatase inhibitors to preserve phosphorylation states

• Fractionation approaches:

  • For analyzing cytoskeletal associations, separate Triton-soluble (cytosolic) and Triton-insoluble (cytoskeletal) fractions

  • MARK2 accumulates in the cytoskeletal fraction in an expression level-dependent manner

• For IHC applications: Use TE buffer pH 9.0 for antigen retrieval, with citrate buffer pH 6.0 as an alternative

These methodological considerations enhance detection specificity and signal strength.

How can researchers investigate MARK2's interactions with cytoskeletal components?

To study MARK2's role in cytoskeletal regulation:

• Co-immunoprecipitation: MARK2 antibodies can be used to isolate protein complexes containing myosin IIA and other interaction partners

• Immunofluorescence: Co-staining MARK2 with paxillin (focal adhesion marker) and actin reveals MARK2 localization to both stress fibers and focal adhesions

• Super-resolution imaging: 3D super-resolution techniques have revealed preferential MARK2 accumulation on dorsal stress fibers

• Biochemical fractionation: Separation of Triton-soluble and -insoluble fractions confirms MARK2 association with the actomyosin cytoskeleton

These complementary approaches provide comprehensive insights into MARK2's multifaceted cytoskeletal roles.

What methodological approaches can assess MARK2's effects on contractility and cell migration?

To analyze MARK2's functional impact on cell behavior:

• Phospho-western blotting: Measure S19-phosphorylated myosin regulatory light chain (pMRLC) in wild-type vs. MARK2-KO cells

• Wound healing assays: Compare migration patterns between control and MARK2-depleted cells

• Kinase assays: In vitro assays with purified MARK2 can determine direct phosphorylation of substrates like MRLC

• Pharmacological inhibition: Combine MARK2 manipulation with ROCK inhibitor (Y27632) and MLCK inhibitor (ML-7) to dissect pathway specificity

Results from these experiments have demonstrated that MARK2 promotes stress fiber formation and activates myosin II in migrating cancer cells through both direct phosphorylation of MRLC and indirect phosphorylation of MYPT1 .

What are common issues with Western blotting using MARK2 antibodies and how can they be resolved?

Common challenges and solutions include:

To optimize signal, researchers should titrate the antibody concentration in each testing system to obtain optimal results .

How can researchers address discrepancies in MARK2 localization across different cell types?

When investigating MARK2 localization:

• Validate with multiple techniques: Combine immunofluorescence, biochemical fractionation, and live-cell imaging

• Consider cell-specific expression patterns: MARK2 shows differential localization based on cell type and state

• Examine multiple fixation methods: Paraformaldehyde, methanol, and glutaraldehyde fixation can reveal different aspects of MARK2 localization

• Control for antibody specificity: Use MARK2-KO cells as negative controls for immunofluorescence studies

Research has demonstrated that MARK2 not only associates with the plasma membrane at cell protrusions and microtubules as previously reported, but also with the actomyosin cytoskeleton and focal adhesions .

How can MARK2 antibodies be used to investigate its role in directional cell migration?

For studying MARK2's role in directional migration:

• Focal adhesion analysis: Co-stain for MARK2 and focal adhesion markers (e.g., paxillin) to analyze focal adhesion formation and orientation

• Membrane domain investigations: Use mutants lacking the membrane binding domain to assess its requirement for focal adhesion targeting and direction-specific protrusions

• FAK phosphorylation studies: Examine whether MARK2 enhances FAK phosphorylation to promote directionally oriented focal adhesions

• Live cell imaging: Track focal adhesion dynamics and cell protrusion in real-time using fluorescently tagged MARK2

These approaches have revealed that MARK2's membrane association is required for focal adhesion targeting, where it specifically enhances cell protrusion by promoting FAK phosphorylation and formation of directionally oriented focal adhesions .

What emerging applications exist for studying MARK2 in immune function?

Recent research has identified MARK2 as a candidate diagnostic gene with immune-associated functions . To investigate this emerging area:

• Immunophenotyping: Compare immune cell populations in wild-type vs. MARK2-deficient models

• Cytokine production: Analyze whether MARK2 modulates inflammatory responses

• Immune cell migration: Assess MARK2's role in immune cell trafficking and tissue infiltration

• Gene expression analysis: Examine co-expression patterns with other immune-related genes like CCDC71, GATA2, and KLRC3

This represents an expanding frontier in MARK2 research beyond its established roles in cell polarity and migration.

What are the optimal storage conditions for maintaining MARK2 antibody activity?

For maximum stability and performance:

• Storage temperature: Store at -20°C, where antibodies remain stable for one year after shipment

• Buffer composition: Most MARK2 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting recommendations: Aliquoting is unnecessary for -20°C storage

• Working solution handling: Once diluted, use within 24 hours and keep at 4°C

Following these storage guidelines ensures consistent antibody performance across experiments.

What considerations should researchers keep in mind when transitioning between applications?

When using MARK2 antibodies across different applications:

• Dilution optimization: Each application requires specific antibody dilutions (see table in section 1.2)

• Buffer compatibility: Ensure buffer components are compatible with the intended application

• Epitope accessibility: Different applications expose epitopes differently; some may require specific antigen retrieval methods

• Species cross-reactivity: Verify antibody performance in the specific species being studied (human, mouse, rat)

Thorough validation in each new experimental system is essential for reliable results.