Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody

What is the Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody and what cellular processes can it help investigate?

The Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody is a polyclonal antibody specifically designed to recognize MARK family proteins (MARK1, MARK2, MARK3, and MARK4) when phosphorylated at threonine 215. This antibody enables researchers to study multiple cellular processes where MARK proteins play crucial roles:

• Cell cycle regulation and mitotic progression

• Cell polarity establishment and maintenance

• Microtubule dynamics and stability

• Cell migration and cytoskeletal organization

• Pathological conditions including cancer and neurodegenerative disorders

The antibody was developed using a synthesized peptide derived from human MARK1/2/3/4 around the phosphorylation site of T215 . Phosphorylation at T215 is known to regulate the activity of MARK family members, influencing their downstream signaling pathways and cellular functions .

What are the recommended applications and dilutions for the Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody?

The Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody has been validated for the following applications with recommended dilutions:

The antibody is provided as a liquid in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide at a concentration of 1 mg/ml . It is recommended to optimize the antibody concentration for each application and experimental system to achieve optimal signal-to-noise ratio.

What species reactivity does this antibody exhibit?

According to product specifications, the Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody shows reactivity towards:

• Human

• Mouse

• Rat

This multi-species reactivity makes the antibody versatile for comparative studies across different model organisms . The conservation of the T215 phosphorylation site across species suggests its functional importance in MARK protein regulation.

What methodologies are recommended for validating the specificity of the Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody?

Validating phospho-specific antibodies is critical for ensuring experimental reliability. Based on current research practices, the following validation methods are recommended:

• Peptide Competition Assay: Incubate the antibody with excess synthetic phosphopeptide corresponding to the T215 region. Loss of signal in subsequent applications confirms epitope specificity .

• Phosphatase Treatment: Treat samples with lambda phosphatase before antibody application. Disappearance of signal confirms phospho-specificity .

• Gene Silencing Approaches: Use RNA interference (siRNA/shRNA) or CRISPR-Cas9 to knock down or knock out MARK genes individually or in combination. Reduced signal in knockdown samples confirms target specificity .

• Molecular Weight Verification: In Western blotting, confirm that detected bands correspond to the expected molecular weights of MARK proteins :

  • MARK1: ~88 kDa

  • MARK2: ~82 kDa

  • MARK3: ~84 kDa

  • MARK4: ~82 kDa

• Phos-tag SDS-PAGE: This technique can separate phosphorylated and non-phosphorylated forms of proteins, allowing verification that the antibody specifically detects the phosphorylated form .

Studies have shown that while phospho-specific antibodies often perform well in Western blotting, they may exhibit cross-reactivity in immunofluorescence or flow cytometry applications where molecular weight discrimination is not possible . Therefore, validation should be performed for each intended application.

How should samples be prepared to ensure optimal detection of phosphorylated MARK proteins?

Proper sample preparation is critical for phosphoprotein detection. The following protocol elements are important:

• Lysis Buffer Composition:

  • Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Use a buffer containing 50 mM HEPES-KOH pH 7.4, 5 mM MgCl₂, 150 mM NaCl, 0.1% NP-40, and 2% glycerol

• Protein Extraction Timing: Rapid extraction is essential to preserve phosphorylation status; process samples immediately after collection.

• Denaturation Conditions: Complete denaturation is necessary to expose phospho-epitopes that may be buried in native protein conformations.

• Protein Quantification: Ensure equal loading by accurate protein quantification methods.

• Gel Electrophoresis:

  • For standard detection: 8-10% SDS-PAGE gels

  • For phospho-isoform separation: 10% SDS-PAGE supplemented with 100 μM Phos-tag and MnCl₂

• Sample Storage: Store samples at -80°C with phosphatase inhibitors to maintain phosphorylation state.

Failure to properly prepare samples may result in false negative results due to dephosphorylation during handling.

How can Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody be used to study the role of MARK family in paclitaxel chemosensitivity?

Recent research has identified MARK2 as a critical regulator of paclitaxel chemosensitivity in pancreatic ductal adenocarcinoma (PDAC). The Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody can be instrumental in elucidating this mechanism through several experimental approaches:

• Monitoring Phosphorylation Dynamics:

  • Track changes in MARK phosphorylation status following paclitaxel treatment

  • Correlate phosphorylation status with cell cycle arrest and apoptotic markers

• Mechanistic Studies:

  • MARK2 is phosphorylated by CDK1 in response to antitubulin chemotherapeutics and during mitosis

  • This phosphorylation is essential for MARK2's role in regulating mitotic progression and paclitaxel cytotoxicity

• MARK2-HDAC-YAP Signaling Axis:

  • MARK2 controls paclitaxel chemosensitivity by directly phosphorylating class IIa HDACs, specifically HDAC4

  • Phosphorylated HDAC4 promotes YAP activation and controls expression of YAP target genes induced by paclitaxel

• Functional Assays:

  • Caspase-Glo 3/7 assay to measure apoptosis after paclitaxel treatment in cells with altered MARK activity

  • Clonogenic assays to assess long-term survival following paclitaxel exposure

Experimental workflow could include:

• Establish MARK2 knockdown cell lines using CRISPR/Cas9

• Compare phospho-MARK status between control and knockdown cells using the T215 antibody

• Treat with paclitaxel and assess effects on cell viability, apoptosis, and colony formation

• Correlate findings with HDAC4 phosphorylation and YAP target gene expression

This approach could identify new strategies for overcoming chemoresistance in PDAC and potentially other cancers.

How does MARK3-mediated phosphorylation couple the actin and microtubule cytoskeletons, and how can this antibody help in studying this mechanism?

MARK3 plays a crucial role in coupling the actin and microtubule cytoskeletons through a phosphorylation-dependent regulatory switch. While the T215 phosphorylation site is not directly involved in this process, monitoring MARK3 activation through T215 phosphorylation can provide insights into this cytoskeletal coupling mechanism:

The mechanism involves:

• MARK3, activated by LKB1, phosphorylates ARHGEF2 (also known as GEF-H1) at Ser151 .

• In its unphosphorylated state, ARHGEF2 is sequestered on microtubules in an inhibited state through binding to dynein light chain Tctex-1 type 1 (DYNLT1) .

• Phosphorylation at Ser151 creates a 14-3-3 binding site on ARHGEF2 .

• The binding of 14-3-3 proteins disrupts the interaction between ARHGEF2 and DYNLT1, causing ARHGEF2 to dissociate from microtubules .

• Released ARHGEF2 becomes activated and stimulates RHOA GTPase activity, promoting formation of stress fibers and focal adhesions .

• This pathway is reversible through PP2A-mediated dephosphorylation of ARHGEF2 at Ser151 .

Research approach using the Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody:

• Correlation Studies:

  • Assess whether T215 phosphorylation correlates with MARK3's ability to phosphorylate ARHGEF2

  • Determine if T215 phosphorylation serves as a biomarker for MARK3 activation

• Kinase Inhibitor Studies:

  • Test whether agents that affect T215 phosphorylation also impact ARHGEF2 phosphorylation and cytoskeletal organization

  • Use the antibody to confirm target engagement in inhibitor studies

• Temporal Signaling Analysis:

  • Establish the sequence of phosphorylation events from MARK activation to cytoskeletal remodeling

  • Determine whether T215 phosphorylation precedes or follows other regulatory phosphorylation events

Understanding this mechanism has implications for cell polarity, migration, and three-dimensional tissue organization, which are crucial processes in development and disease.

What are the best practices for quantifying phosphorylation levels using Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody in phospho-protein arrays?

Phospho-protein arrays offer a high-throughput approach for analyzing multiple phosphorylation events simultaneously. When using the Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody in such arrays, several methodological considerations should be addressed:

• Array Platform Selection:

  • Reverse Phase Protein Arrays (RPPA) allow screening of multiple samples against the T215 antibody

  • Antibody arrays permit detection of T215 phosphorylation alongside other phosphorylation events

• Data Acquisition Protocol:

  • Nitrocellulose membranes should include specific antibodies spotted in duplicate

  • Include positive reference double spots and PBS-only negative controls

  • For phospho-RTK arrays, use pan-anti-phospho-tyrosine antibodies conjugated with HRP

  • For phospho-MAPK arrays, use biotinylated anti-phospho-kinase antibodies followed by streptavidin-HRP

• Normalization Strategy:

  • Normalize phospho-specific signals to total MARK protein levels to account for expression differences

  • This correction helps account for loading errors and gel-to-gel variations (standard deviation typically within 6% of mean)

• Quality Control Measures:

  • AP-treatment induced logFC value can serve as a predictor of antibody quality

  • A logFC cut-off value of -0.792 has demonstrated good predictive ability for phospho-antibody performance (area under ROC curve of 0.825)

  • Verify key findings from arrays using Western blot analysis

• Quantification Approach:

  • Use appropriate image analysis software with background subtraction

  • Apply log-log plots to assess linearity of antibody response

  • Consider statistical methods that account for variability in antibody performance

• Validation:

  • Approximately 85% of antibodies selected based on the logFC criterion show meaningful single bands at expected sizes in Western blot validation

  • Consider complementary methods like mass spectrometry for critical phosphorylation sites

Comparative studies have shown that different quantification methods may yield varying results. Mass spectrometry and NMR typically show excellent agreement with each other but can show significant discrepancies compared to antibody-based methods like Western blots .

What are the potential cross-reactivity issues with Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody and how can they be mitigated?

Phospho-specific antibodies can exhibit various cross-reactivity issues that researchers should be aware of and address:

Potential Issues:

• Recognition of Similar Phosphomotifs:

  • The antibody may recognize similar phosphorylation motifs in unrelated proteins

  • This is particularly pronounced during mitosis when numerous proteins are phosphorylated

• Application-Specific Cross-Reactivity:

  • An antibody may perform well in Western blotting but show cross-reactivity in immunofluorescence

  • Studies have shown that phospho-specific antibodies can associate with spindle poles in mitotic cells through binding to unidentified phosphoproteins

• Nonspecific Bands:

  • Additional bands may appear, particularly in samples treated with agents affecting microtubule dynamics like nocodazole

  • These can often be distinguished from MARK proteins by their molecular weights

• Tissue Fixation Effects:

  • Formalin fixation can affect phospho-epitope detection, with varying correlation between FFPE and fresh-frozen samples

Mitigation Strategies:

• Validation Controls:

  • Include positive controls (known high phospho-MARK samples)

  • Include negative controls (phosphatase-treated samples)

  • Use samples with MARK genes knocked down or knocked out

• Application-Specific Controls:

  • For immunofluorescence: Include peptide competition controls and secondary-only controls

  • For flow cytometry: Use isotype controls and phosphatase-treated samples

• Complementary Detection Methods:

  • Verify key findings with orthogonal techniques like mass spectrometry

  • Use Phos-tag SDS-PAGE to separate phospho-isoforms

• Sample Preparation Refinements:

  • Include comprehensive phosphatase inhibitor cocktails

  • Optimize protein extraction and denaturation conditions

  • Consider synchronizing cells to minimize cell cycle-dependent variability

• Data Analysis Approaches:

  • Normalize phospho-specific signals to total protein levels

  • Compare patterns across multiple antibodies targeting different epitopes of the same protein

Implementing these mitigation strategies will help ensure more reliable and interpretable data when using the Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody in various experimental settings.

How can researchers differentiate between the activities of different MARK isoforms using this antibody?

• Sequential Immunoprecipitation:

  • First immunoprecipitate with isoform-specific antibodies against MARK1, MARK2, MARK3, or MARK4

  • Then probe immunoprecipitates with the Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody

  • Quantify the relative phosphorylation of each isoform

• Isoform-Specific Knockdown/Knockout:

  • Generate cells with individual MARK isoforms knocked down or knocked out

  • Analyze changes in T215 phosphorylation signal to determine each isoform's contribution

  • Example method: "To construct the EGFP-expressing all-in-one CRISPR/Cas9n plasmid targeting human MARK2, the sense and antisense oligonucleotides were synthesized, annealed and Golden Gate-assembled"

• Expression of Tagged Variants:

  • Express tagged versions of each MARK isoform in cells with endogenous MARK proteins depleted

  • Use tag-specific antibodies to pull down individual isoforms

  • Analyze T215 phosphorylation status for each tagged isoform

• Substrate-Specific Analysis:

  • Different MARK isoforms have preferential substrates:

    • MARK3 phosphorylates ARHGEF2 at Ser151

    • MARK2 phosphorylates HDAC4 during antitubulin treatment

  • Correlate T215 phosphorylation with these isoform-specific substrate phosphorylation events

• Subcellular Localization:

  • MARK isoforms may exhibit different subcellular distributions

  • Combine the T215 antibody with isoform-specific antibodies in immunofluorescence studies

  • Analyze colocalization patterns to infer which isoforms are phosphorylated in specific cellular compartments

• In Vitro Kinase Assays:

  • Use recombinant MARK isoforms in kinase assays with known substrates

  • Include [γ-³²P]ATP and analyze phosphorylation by autoradiography

  • Correlate substrate phosphorylation with T215 phosphorylation detected by the antibody

By integrating these approaches, researchers can gain insights into the relative contributions and specific roles of individual MARK isoforms despite using an antibody that recognizes a conserved phosphorylation site.

How can Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody be incorporated into studies of kinase activity inference using functional networks?

Incorporating the Phospho-MARK1/MARK2/MARK3/MARK4 (T215) Antibody into kinase activity inference studies enables researchers to understand how MARK proteins integrate into broader signaling networks. This approach combines experimental data with computational methods to infer kinase activities from phosphoproteomic datasets.

Methodological Framework:

• Experimental Data Generation:

  • Use the T215 antibody to detect MARK phosphorylation across various experimental conditions

  • Generate phosphoproteomics data from perturbation studies (e.g., kinase inhibitors, growth factors)

  • Apply quality control steps including:

    • Restricting analysis to mono-phosphorylated peptides mapped to canonical transcripts

    • Averaging log-fold changes of technical replicates

    • Filtering out peptides identified in only a single study

• Integration with Known Kinase-Substrate Networks:

  • Combine antibody-derived data with existing knowledge from databases like PhosphositePlus

  • PhosphositePlus contains 10,476 kinase-substrate links for 371 distinct kinases and 7,480 sites

  • Map T215 phosphorylation site to these networks to understand its position in signaling cascades

• Benchmarking Activity Inference Methods:

  • Evaluate accuracy using metrics like P_hit(k) ("top-k-hit")

  • This metric calculates percentage of perturbations where the inference method successfully identifies true perturbed kinases among top k predicted kinases

  • Use the T215 antibody data as part of gold standard validation datasets

• Multi-Kinase Analysis:

  • Design arrays containing antibodies against key kinases in the MARK signaling network

  • Use standard phospho-protein array protocols:

    • Spot specific antibodies in duplicate on nitrocellulose membranes

    • Include positive reference spots and negative controls

    • Apply tissue lysates and detect with appropriate secondary reagents

• Network Construction and Visualization:

  • Build functional networks incorporating both direct and indirect interactions

  • Weight network edges based on experimental confidence

  • Visualize MARK phosphorylation status in the context of upstream and downstream signaling events

Analysis Considerations:

• Data Processing Pipeline:

  • For quantitative analysis, normalize phospho-specific signal to total protein signal

  • Apply appropriate statistical methods to handle missing values and technical variation

  • Consider both absolute phosphorylation levels and relative changes across conditions

• Algorithm Selection:

  • Different algorithms may perform better for different kinase families

  • Evaluate multiple methods for inferring MARK activity

  • Consider algorithms that can incorporate prior knowledge about kinase-substrate relationships

• Biological Interpretation:

  • Correlate inferred MARK activity patterns with biological phenotypes

  • Look for context-specific patterns of MARK activation

  • Generate hypotheses about novel MARK functions based on network analysis

This integrated approach can reveal how MARK family kinases function within signaling networks and how their activities coordinate with other kinases to regulate cellular processes such as cell polarity, migration, and cell cycle progression.