Phospho-MARK2 (T596) Antibody

What is MARK2 and why is phosphorylation at T596 significant?

MARK2 (also known as PAR-1) is a member of the Par-1 family of serine/threonine protein kinases. It functions as an important regulator of cell polarity in epithelial and neuronal cells, and controls microtubule stability through phosphorylation and inactivation of several microtubule-associating proteins . MARK2 localizes to cell membranes and exists in multiple isoforms resulting from alternative splicing .

Phosphorylation at T596 (sometimes referred to as T595 depending on the isoform) represents a regulatory modification that affects MARK2 activity. This specific phosphorylation site is critical for modulating MARK2's kinase function, influencing its role in microtubule dynamics, cellular polarization, and potentially in pathways related to cancer, immunology, and nuclear signaling . The phosphorylation status at this residue serves as an important indicator of MARK2 activation state in experimental systems.

What applications are Phospho-MARK2 (T596) antibodies suitable for?

Phospho-MARK2 (T596) antibodies have been validated for several common laboratory applications:

These antibodies are particularly valuable for research focusing on cancer mechanisms, immunological processes, and nuclear signaling pathways where MARK2 plays important regulatory roles . When selecting an application, it's important to validate the antibody in your specific experimental system, as performance can vary depending on tissue type, fixation methods, and other experimental conditions.

How should Phospho-MARK2 (T596) antibodies be stored and handled?

Proper storage and handling of Phospho-MARK2 (T596) antibodies is essential for maintaining their specificity and sensitivity. Based on manufacturer recommendations:

Store unopened antibody vials at -20°C prior to opening . After initial use, it's advisable to make small aliquots and freeze them at -20°C or below for extended storage to avoid repeated freeze-thaw cycles which can degrade antibody quality . The antibody solution may be kept at 4°C for several weeks as an undiluted liquid if in continuous use .

When working with the antibody, centrifuge the product if it's not completely clear after standing at room temperature . Dilute only immediately before use to maintain optimal antibody concentration and performance . The typical working solution contains 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2, with 0.01% (w/v) Sodium Azide as a preservative .

The expiration date is typically one year from the date of opening when stored properly, though this may vary between manufacturers .

What is the difference between antibodies targeting T595 versus T596 phosphorylation sites?

The numerical designation of the phosphorylation site (T595 versus T596) often reflects different isoforms of MARK2 rather than distinct phosphorylation sites. MARK2 exists in multiple isoforms due to alternative splicing, with some containing a 162-bp alternative exon that results in longer protein variants .

The Boster Bio antibody is designated as Anti-Phospho-MARK2 T595, specifically detecting human MARK2 isoform a when phosphorylated at threonine 595 . Meanwhile, other antibodies may be labeled as targeting T596, such as the antibody described in search result , which detects endogenous levels of MARK2 protein only when phosphorylated at T596 .

Despite the different numbering, these antibodies target functionally equivalent phosphorylation sites. When selecting an antibody, researchers should carefully review the product information to ensure the antibody recognizes the specific isoform and phosphorylation site relevant to their research question, and validate the antibody in their experimental system.

How can I validate the specificity of a Phospho-MARK2 (T596) antibody?

Rigorous validation of Phospho-MARK2 (T596) antibody specificity is crucial for ensuring reliable experimental results. A comprehensive validation approach should include multiple complementary methods:

CRISPR/Cas9 Knockout Validation: Generate MARK2 knockout cell lines using CRISPR/Cas9 technology in cells with high endogenous MARK2 expression. Compare antibody signals between parental and knockout cell lines using Western blotting, which should show absence of signal in the knockout line if the antibody is specific .

Phosphatase Treatment Controls: Treat cell lysates with lambda phosphatase to remove phosphorylation and compare with untreated samples. A phospho-specific antibody should show significantly reduced or absent signal in phosphatase-treated samples .

Phospho-Blocking Peptide: Perform competition assays using the phosphorylated peptide immunogen. Pre-incubating the antibody with the phosphorylated peptide should block specific binding, while pre-incubation with non-phosphorylated peptide should not affect signal if the antibody is truly phospho-specific .

Mosaic Immunofluorescence: For IF applications, create a mosaic culture containing both wildtype and MARK2 knockout cells on the same coverslip (distinguishable by expression of different fluorescent markers), then perform immunostaining. This allows direct comparison of staining pattern in the presence and absence of the target protein under identical conditions .

Implementation of such validation steps addresses the reproducibility crisis resulting from non-specific antibodies and ensures high confidence in subsequent experimental results.

How does cell fixation method affect Phospho-MARK2 (T596) antibody performance in immunofluorescence?

The choice of fixation method can significantly impact the performance of phospho-specific antibodies in immunofluorescence applications, including Phospho-MARK2 (T596) antibodies. Different fixation approaches preserve protein structures and epitopes differently, affecting antibody binding and signal quality.

Methanol Fixation: Cold methanol fixation (chilled at -20°C for 10 minutes) often provides better exposure of phospho-epitopes by precipitating proteins rather than cross-linking them . This approach can be particularly beneficial for phospho-specific antibodies as it often preserves phosphorylation sites while removing lipids, which can improve antibody penetration.

Comparative Approach: For optimal results, testing both fixation methods in parallel is recommended. As noted in search result , researchers examined antibody performance with both PFA and methanol fixation methods in their validation pipeline. A side-by-side comparison allows identification of the method that provides the best signal-to-noise ratio for the specific phospho-epitope.

After fixation, optimal immunofluorescence protocol includes blocking and permeabilization in buffer containing TBS, 5% BSA and 0.3% Triton X-100 (pH 7.4) for 1 hour at room temperature, followed by overnight incubation with the primary antibody at 4°C (typically at 2 μg/ml concentration) .

How can I troubleshoot weak or non-specific signals when using Phospho-MARK2 (T596) antibody?

When encountering weak or non-specific signals with Phospho-MARK2 (T596) antibody, systematic troubleshooting can help identify and resolve the issues:

For Weak Signals:

• Cell Line Selection: Screen multiple cell lines to identify those with higher endogenous expression of MARK2. Consulting protein expression databases like PaxDb can provide initial guidance, but direct validation through immunoblotting is essential as database predictions may not always match actual expression levels .

• Phosphorylation Status: Phosphorylation at T596 may be transient or stimulus-dependent. Consider treating cells with phosphatase inhibitors (e.g., okadaic acid, calyculin A) to preserve phosphorylation, or use appropriate stimuli to induce the phosphorylation event you're studying.

• Antibody Concentration: Increase antibody concentration incrementally, starting from the manufacturer's recommended dilution. For Western blotting, try 1:500 instead of 1:1000; for IF, start with 1:200 and adjust as needed .

• Signal Enhancement: For Western blotting, consider using more sensitive detection methods like ECL-Prime or Odyssey infrared detection systems, which can detect lower abundance phospho-proteins .

For Non-specific Signals:

• Blocking Optimization: Increase blocking stringency by using 5% BSA in TBST for Western blots and 5% BSA with 0.3% Triton X-100 for immunofluorescence . Longer blocking times (2 hours instead of 1 hour) may help reduce background.

• Validation Controls: Always include a negative control (MARK2 knockout or siRNA-treated cells) to distinguish between specific and non-specific bands or staining patterns .

• Cross-Reactivity Testing: Test antibody against cell lines expressing related kinase family members to ensure specificity. This is particularly important for MARK family antibodies due to sequence similarity between MARK1-4.

• Gradient Gels: For Western blotting, use 5-16% gradient polyacrylamide gels to achieve better separation of proteins with similar molecular weights, helping distinguish specific bands from non-specific signals .

Systematic application of these troubleshooting approaches should help optimize experimental conditions for reliable detection of phosphorylated MARK2.

How does phosphorylation at T596 relate to MARK2's cellular functions?

Phosphorylation at T596 represents a key regulatory mechanism affecting MARK2's diverse cellular functions, which include:

Microtubule Regulation: MARK2 controls microtubule stability through phosphorylation of microtubule-associated proteins (MAPs) . Phosphorylation at T596 may modulate MARK2's ability to phosphorylate these substrates, thereby affecting microtubule dynamics in processes such as cell division, migration, and intracellular transport.

Cell Polarity Establishment: As a member of the Par-1 family, MARK2 is a critical regulator of cell polarity in epithelial and neuronal cells . T596 phosphorylation likely influences MARK2's interaction with polarity complexes and its subcellular localization, affecting asymmetric cell division, directional migration, and epithelial cell organization.

Membrane Localization: MARK2 localizes to cell membranes, and its proper localization is essential for its function . Phosphorylation at T596 may regulate this membrane association, potentially through interaction with scaffold proteins or other membrane-associated factors.

Immune Response Regulation: MARK2 has been implicated in immunological processes . The phosphorylation status at T596 may influence these functions, potentially modulating immune cell activation, migration, or signaling.

Phagosome/Lysosome Interactions: Drawing parallels from studies of other disease-related proteins like C9ORF72, which localizes to phagosomes/lysosomes , MARK2 may have functions related to vesicular trafficking or autophagy that are regulated by T596 phosphorylation.

Understanding how T596 phosphorylation affects these functions requires careful experimental approaches, including the use of phospho-mimetic and phospho-deficient mutants, temporal analysis of phosphorylation during cellular processes, and identification of kinases and phosphatases that regulate this specific site.

What are the optimal cell lines for studying Phospho-MARK2 (T596) in different research contexts?

Selecting appropriate cell lines with significant MARK2 expression is crucial for successful experiments with Phospho-MARK2 (T596) antibodies. Based on the available information and broader research practices:

When selecting cell lines, consider these methodological approaches:

• Expression Database Verification: Consult protein expression databases like PaxDb for initial guidance, but always verify with direct testing as database predictions may not always align with actual expression (as noted with RKO cells in the search results) .

• Knockout Generation Strategy: For validation studies, prioritize generating CRISPR/Cas9 knockouts in the cell line with highest endogenous expression rather than multiple lines with varying expression. For MARK2, U2OS cells have proven effective for this purpose .

• Cell-Type Specific Functions: Consider the biological context of your research question. For studying MARK2 in neuronal polarity, neuronal cell lines or primary neurons might be more relevant despite potentially lower expression levels. For immune functions, consider macrophage or lymphocyte cell lines.

• Transfection Efficiency: For overexpression studies, cell lines with high transfection efficiency like HEK-293 may be preferable, even if their endogenous MARK2 expression is not the highest .

• Phosphorylation Status: Different cell lines may exhibit varying levels of T596 phosphorylation based on their signaling contexts. Screening multiple cell lines specifically for the phosphorylated form may reveal different optimal choices than screening for total MARK2.

The optimal approach involves initial screening of multiple cell lines with validated antibodies, followed by selection based on both expression level and relevance to the biological question being investigated.

What is the most effective immunoprecipitation protocol for Phospho-MARK2 (T596) antibody?

An effective immunoprecipitation (IP) protocol for Phospho-MARK2 (T596) antibody requires careful optimization to preserve phosphorylation status while achieving specific pulldown. Based on the search results and standard practices for phospho-proteins:

Cell Lysis Buffer Composition:

• 50 mM Tris-HCl (pH 7.5)

• 150 mM NaCl

• 1% NP-40 or Triton X-100

• 0.5% sodium deoxycholate

• Phosphatase inhibitor cocktail (critical for preserving phosphorylation)

• Protease inhibitor cocktail

• 5 mM EDTA

• 5 mM EGTA

Detailed Protocol:

• Cell Preparation: Culture cells to approximately 80-90% confluence in 10 cm dishes. Prior to lysis, consider treating cells with phosphatase inhibitors like okadaic acid or calyculin A to enhance phosphorylation signal.

• Lysis Procedure: Wash cells twice with ice-cold PBS, then add 1 mL of ice-cold lysis buffer directly to the plate. Scrape cells and transfer to microcentrifuge tubes. Incubate on ice for 30 minutes with occasional gentle vortexing.

• Lysate Clarification: Centrifuge at 14,000 g for 15 minutes at 4°C. Transfer supernatant to new tube and determine protein concentration.

• Pre-clearing: Add 50 μL of Protein A/G beads to 1 mg of protein lysate and rotate for 1 hour at 4°C to reduce non-specific binding. Centrifuge at 1,000 g for 5 minutes and transfer supernatant to new tube.

• Antibody Binding: Add 5-10 μg of Phospho-MARK2 (T596) antibody to the pre-cleared lysate. Incubate with gentle rotation overnight at 4°C. In parallel, prepare a negative control using non-immune IgG from the same species as the antibody.

• Immunoprecipitation: Add 50 μL of fresh Protein A/G beads to the antibody-lysate mixture and incubate with rotation for 4 hours at 4°C.

• Washing: Pellet beads by centrifugation at 1,000 g for 5 minutes. Wash beads 3 times with lysis buffer containing phosphatase inhibitors, followed by 2 washes with TBS (12 mM NaHCO3, 0.1 mM EDTA) .

• Elution: Elute bound proteins by adding 50 μL of 2X SDS sample buffer and boiling for 5 minutes. Centrifuge at 1,000 g for 5 minutes and collect supernatant for SDS-PAGE analysis.

The resulting immunoprecipitated material can be analyzed by immunoblotting with either the same antibody or another antibody against MARK2 to confirm specificity, or with antibodies against potential interacting partners to study protein-protein interactions.

How should quantitative Western blot analysis be performed with Phospho-MARK2 (T596) antibody?

Quantitative Western blot analysis with Phospho-MARK2 (T596) antibody requires rigorous methodology to ensure accurate and reproducible results, particularly when assessing changes in phosphorylation levels:

Sample Preparation and Protein Normalization:

• Extract proteins using a buffer containing phosphatase inhibitors to preserve phosphorylation status.

• Determine protein concentration using a reliable method like BCA assay.

• Load equal amounts of protein per lane (typically 20-50 μg).

• Include a total protein stain (like REVERT) for normalization, which is more reliable than single housekeeping protein controls .

Electrophoresis and Transfer:

• Use large format 5-16% gradient polyacrylamide gels for optimal separation of the 87 kDa MARK2 protein .

• Transfer to nitrocellulose membranes (preferred over PVDF for quantitative analysis).

• Verify transfer efficiency with reversible total protein stains like Ponceau S .

Antibody Incubation:

• Block membranes with 5% milk in TBST.

• Incubate with Phospho-MARK2 (T596) antibody (1:1000 dilution) in 5% BSA in TBST overnight at 4°C .

• For fluorescent detection, wash thoroughly and incubate with IRDye-conjugated secondary antibody (1:20,000) in 5% BSA in TBST for 1 hour at room temperature .

Detection and Analysis:

• For chemiluminescence: Use high-sensitivity ECL reagents and calibrated imaging systems with extended dynamic range.

• For fluorescence: Use an infrared imaging system like LI-COR Odyssey for superior quantification .

• Analyze band intensities using dedicated software (e.g., LI-COR Image Studio Lite) .

• Normalize phospho-MARK2 signal to total protein loading using the total protein stain.

• For relative phosphorylation levels, probe duplicate membranes with total MARK2 antibody and calculate phospho-MARK2/total MARK2 ratio.

Controls and Validation:

• Include positive control (cells treated with agents known to induce MARK2 phosphorylation).

• Include negative control (MARK2 knockout cells) .

• Include phosphatase-treated lysate as negative control for phospho-specific detection.

• Perform technical replicates (minimum 3) and biological replicates (minimum 3) for statistical analysis.

This comprehensive approach ensures quantitative data that accurately reflects the phosphorylation status of MARK2 at T596 under different experimental conditions.

How does Phospho-MARK2 (T596) antibody performance compare across different commercial sources?

When selecting a Phospho-MARK2 (T596) antibody for research, understanding performance differences between commercial sources is crucial. Based on the search results and general antibody validation principles:

Key Performance Considerations:

• Epitope Specificity: Both antibodies target similar phosphorylation sites (T595/T596), but the specific synthetic peptide immunogen sequence may differ between manufacturers, potentially affecting detection of different MARK2 isoforms .

• Validation Rigor: Consider manufacturers that provide comprehensive validation data, including positive/negative controls and specificity demonstrations using techniques like CRISPR/Cas9 knockouts . The reproducibility crisis in antibody research emphasizes the importance of rigorous validation evidence .

• Lot-to-Lot Consistency: Polyclonal antibodies may show variation between production lots. Some manufacturers provide lot-specific validation data or maintain consistent polyclonal pools to minimize this variation .

• Cross-Reactivity Testing: Evaluate whether the manufacturer has tested for cross-reactivity with other MARK family members (MARK1, MARK3, MARK4) which share sequence similarity .

• Application-Specific Performance: An antibody might perform well in Western blot but poorly in immunofluorescence, or vice versa. Select based on your primary application needs .

When switching between antibody sources or lots, it's advisable to perform parallel validation experiments to ensure comparable performance. The implementation of standardized validation procedures, as described in search result , can help address the reproducibility challenges associated with antibody-based research.

How can Phospho-MARK2 (T596) antibody be used in combination with other tools to study MARK2 signaling pathways?

Integrating Phospho-MARK2 (T596) antibody with complementary research tools creates a powerful approach for comprehensive analysis of MARK2 signaling pathways:

Multi-Antibody Analysis:

• Phosphorylation Dynamics: Use antibodies against different MARK2 phosphorylation sites (not just T596) to map phosphorylation patterns in response to various stimuli.

• Total vs. Phosphorylated Protein: Combine phospho-specific antibody with total MARK2 antibody to determine the proportion of phosphorylated protein under different conditions .

• Signaling Cascade Analysis: Simultaneously probe for upstream kinases (like LKB1, TAO kinases) and downstream substrates (such as Tau, MAP2, or MAPT) to map the complete signaling axis.

Genetic Manipulation Approaches:

• CRISPR/Cas9 Systems: Generate knockout cell lines for validation, but also create knock-in cell lines expressing T596A (phospho-deficient) or T596E (phospho-mimetic) mutants to study functional consequences of this phosphorylation .

• Inducible Expression Systems: Develop Tet-on/off systems expressing wildtype or mutant MARK2 in knockout backgrounds to control MARK2 activity temporally.

• siRNA/shRNA: Use RNA interference to achieve transient or stable knockdown as complementary approaches to CRISPR-based methods .

Advanced Imaging Techniques:

• Co-localization Studies: Combine Phospho-MARK2 (T596) antibody with markers for specific cellular compartments (e.g., LAMP1-YFP for lysosomes) to determine phosphorylation-dependent localization patterns .

• FRET Biosensors: Develop FRET-based reporters to monitor MARK2 activation or T596 phosphorylation in real-time in living cells.

• Super-Resolution Microscopy: Apply techniques like STORM or PALM with validated phospho-antibodies to visualize nanoscale spatial organization of phosphorylated MARK2.

Proteomic and Phosphoproteomic Analysis:

• Phospho-Enrichment + Mass Spectrometry: Combine immunoprecipitation using the Phospho-MARK2 (T596) antibody with mass spectrometry to identify interacting partners specific to the phosphorylated form.

• Temporal Phosphoproteomics: Use antibody-based enrichment followed by phosphoproteomic analysis to identify downstream substrates affected by MARK2 T596 phosphorylation status.

This integrated approach provides a comprehensive understanding of how T596 phosphorylation regulates MARK2 function within complex cellular signaling networks.

What are the current limitations in Phospho-MARK2 (T596) antibody technology and future directions?

Current limitations in Phospho-MARK2 (T596) antibody technology highlight challenges that researchers should consider, while also pointing to future developments that may enhance their utility:

Current Limitations:

• Isoform Specificity: Most current antibodies don't clearly distinguish between different MARK2 splice variants, which may have distinct functions and regulation patterns . This limits our understanding of isoform-specific phosphorylation events.

• Cross-Reactivity Concerns: The high sequence homology between MARK family members (MARK1-4) creates potential for cross-reactivity that may not be fully characterized for all commercial antibodies .

• Dynamic Range Limitations: Current antibody-based detection methods may not capture the full dynamic range of phosphorylation changes, particularly for low-abundance or transiently phosphorylated pools of MARK2.

• Reproducibility Issues: The antibody reproducibility crisis affects all research antibodies, including phospho-specific ones, necessitating rigorous validation for each new lot or application .

• Context-Dependent Phosphorylation: The T596 phosphorylation may be regulated differently across cell types and conditions, making it challenging to standardize protocols across research contexts.

Future Directions:

• Recombinant Antibody Technology: Development of recombinant monoclonal antibodies with defined specificity profiles would address lot-to-lot variability issues that affect polyclonal antibodies .

• Isoform-Specific Antibodies: Design of antibodies that can distinguish between different MARK2 splice variants while still recognizing the phosphorylated T596 residue.

• Multiplexed Detection Systems: Advanced multiplexing technologies to simultaneously monitor multiple phosphorylation sites on MARK2 and related signaling molecules in the same sample.

• Synthetic Biology Approaches: Development of engineered cellular reporters that can monitor T596 phosphorylation in real-time without requiring fixation or cell lysis.

• Community Standards: Implementation of standardized validation protocols across the research community, as proposed in search result , would significantly enhance confidence in antibody-based findings.

• Therapeutic Applications: As MARK2 signaling becomes better understood, development of antibodies that can modulate its activity through specific binding to phosphorylated forms might have therapeutic potential in diseases where MARK2 signaling is dysregulated.

Addressing these limitations while pursuing these future directions will strengthen the foundation of MARK2 research and contribute to more reliable and informative studies of this important kinase in various physiological and pathological contexts.

How can researchers contribute to improving antibody validation standards for Phospho-MARK2 and other phospho-specific antibodies?

Researchers can play a crucial role in advancing antibody validation standards for phospho-specific antibodies like Phospho-MARK2 (T596) through several actionable approaches:

Implement Comprehensive Validation Protocols:

• Adopt the Knockout-Based Validation Pipeline: Follow the example described in search result , which uses CRISPR/Cas9 to generate knockout cell lines for definitive antibody validation. For phospho-specific antibodies, this should be coupled with phosphatase treatments to confirm phospho-specificity.

• Document Multiple Applications: Validate each antibody across multiple applications (WB, IF, IP) rather than just the primary intended use, and publish detailed protocols for each successful application .

• Publish Negative Results: Share information about antibodies that fail validation tests to prevent other researchers from encountering the same issues, potentially through platforms like Antibodypedia or Research Resource Identifiers (RRIDs).

Enhance Reporting and Reproducibility:

• Standardize Reporting: Always include complete antibody information in publications (manufacturer, catalog number, lot number, validation methods, dilutions, incubation conditions) .

• Share Raw Data: Provide unedited blot images and immunofluorescence micrographs as supplementary material, including both positive and negative controls.

• Deposit Validation Protocols: Contribute detailed validation protocols to repositories like protocols.io to facilitate methodology sharing.

Engage with Community Initiatives:

• Participate in Validation Consortia: Join collaborative efforts like the Antibody Validation Initiative or contribute to databases like Antibodypedia that aim to standardize antibody validation.

• Advocate for Journal Standards: Support journals that require rigorous antibody validation data and encourage others to adopt similar standards.

• Develop Field-Specific Guidelines: Collaborate with colleagues to establish phospho-antibody-specific validation standards that address the unique challenges of these reagents.

Advance Technology and Methods:

• Generate Community Resources: Create and share MARK2 knockout cell lines or recombinant MARK2 protein standards that can serve as controls for the wider research community .

• Explore Alternative Approaches: Develop and validate complementary methods for detecting phosphorylated MARK2, such as proximity ligation assays or targeted mass spectrometry, to provide orthogonal validation of antibody-based results.

• Engage Commercial Providers: Provide feedback to antibody manufacturers about performance and collaborate on improved validation standards.