MASTL Antibody

What is MASTL and why is it important in cell biology research?

MASTL (Microtubule Associated serine/threonine Kinase-Like) is an essential kinase that plays a critical role in cell cycle regulation. Its primary function involves preventing early dephosphorylation of M-phase targets of Cdk1/CycB by inhibiting the activity of the PP2A-B55δ phosphatase complex . MASTL achieves this inhibition indirectly by phosphorylating two related paralogs, Arpp19 and ENSA, which then inhibit PP2A-B55δ . What makes MASTL particularly unique is its structure - it contains a non-conserved insertion of 550 residues within its activation loop, splitting the kinase domain into two parts . This unusual structure contributes to MASTL's target specificity and activity regulation. Beyond cell cycle control, recent studies have identified MASTL's involvement in cancer progression, DNA damage response, and regulation of cytoskeletal dynamics in platelets, making it a versatile research target across multiple biological disciplines .

Which applications are most commonly used with MASTL antibodies?

MASTL antibodies are versatile tools employed across multiple experimental applications in molecular and cellular biology research. The most commonly used applications include:

• Western Blotting (WB): For detecting endogenous levels of total MASTL protein in cell or tissue lysates, allowing quantification of expression levels

• Immunofluorescence (IF)/Immunocytochemistry (ICC): For visualizing subcellular localization of MASTL in fixed cells, often used to track its distribution during different cell cycle stages

• Immunohistochemistry (IHC): For detecting MASTL expression in tissue sections, particularly useful in cancer research to correlate expression with clinical parameters

• ELISA: For quantitative measurement of MASTL protein levels

• Immunoprecipitation (IP): For isolating MASTL protein complexes to study protein-protein interactions and post-translational modifications

The selection of the appropriate application depends on the specific research question, with most commercial MASTL antibodies validated for multiple applications to provide flexibility in experimental design .

How do I select the appropriate MASTL antibody for my research?

Selecting the appropriate MASTL antibody requires careful consideration of several factors to ensure experimental success. First, identify your target region of interest within the MASTL protein. Some antibodies target the C-terminal region , while others recognize the N-terminal portion or specific internal epitopes . This distinction is critical if you're studying particular MASTL domains or if post-translational modifications might mask certain epitopes.

Second, consider antibody specificity and cross-reactivity. Review the predicted reactivity information to ensure the antibody will recognize your species of interest. Many MASTL antibodies detect human and mouse proteins, but cross-reactivity with other species varies . For evolutionary studies, antibodies with broad cross-reactivity across species (pig, zebrafish, bovine, etc.) may be advantageous .

Third, match the antibody format to your application. For Western blotting and immunoprecipitation, both polyclonal and monoclonal antibodies can work effectively, though monoclonals often provide higher specificity. For immunofluorescence applications, confirmed IF-validated antibodies are essential . Finally, verify that the antibody has been validated specifically for your application through published literature or manufacturer validation data to minimize experimental troubleshooting time .

What controls should I include when using MASTL antibodies?

When designing experiments with MASTL antibodies, proper controls are essential to ensure reliable and interpretable results. For primary validation, include both positive and negative controls:

• Positive controls: Use cell lines or tissues known to express high levels of MASTL, such as proliferating cancer cells (particularly ER-negative, HER2-negative breast cancer cells like MDA-MB-231), which have been documented to express significant amounts of MASTL .

• Negative controls: Include MASTL-knockdown or knockout samples generated through siRNA, shRNA, or CRISPR-Cas9 approaches to confirm antibody specificity . The search results mention specific MASTL-silencing approaches with inducible shRNAs and siRNAs that can be replicated .

• Secondary antibody-only controls: To rule out non-specific binding of secondary antibodies, especially crucial for immunofluorescence and immunohistochemistry applications.

• Loading controls: For Western blotting, include appropriate loading controls such as α-tubulin to normalize protein amounts across samples.

• Peptide competition assays: Where the antibody is pre-incubated with the immunizing peptide before application, which should abolish specific binding if the antibody is truly specific .

These controls help validate antibody specificity and provide critical reference points for interpreting experimental results, particularly when examining MASTL expression or phosphorylation changes under different experimental conditions .

How does MASTL function differ between mitotic regulation and cancer progression?

MASTL's role extends significantly beyond its canonical mitotic function, with distinct mechanisms in cancer progression. In mitosis, MASTL primarily acts through a well-defined pathway: it phosphorylates substrates Arpp19 and ENSA, which subsequently inhibit PP2A-B55δ phosphatase activity, preventing premature dephosphorylation of CDK1 substrates and ensuring proper mitotic progression . This mechanism is tightly regulated through precise activation and inactivation timing to maintain cellular integrity during division.

Furthermore, MASTL supports TGF-β signaling in cancer cells by maintaining TGFBR2 (TGF-β receptor II) expression and promoting activation of downstream SMAD3 and AKT pathways . This represents a novel non-mitotic function that potentially contributes to cancer cell plasticity, survival, and metastatic capacity. These cancer-specific functions make MASTL a promising therapeutic target, as its inhibition could simultaneously disrupt both cell cycle progression and stemness maintenance in cancer cells .

How does the thrombocytopenia-associated mutation affect MASTL function at the molecular level?

The thrombocytopenia-associated mutation in MASTL produces profound effects on protein function that extend beyond simple loss or gain of activity. Research using knock-in mouse models (MastlED/ED) has revealed that this mutation represents a pathogenic dominant mutation that fundamentally alters MASTL's regulatory relationship with PP2A phosphatase .

At the molecular level, the mutation appears to mimic decreased PP2A activity, resulting in altered phosphorylation patterns of cytoskeletal regulatory pathways. Unlike MASTL deficiency (MastlΔ/Δ) which prevents proper megakaryocyte maturation, the thrombocytopenia-associated mutation leads to aberrant activation and reduced survival of platelets . This suggests a gain-of-function effect rather than simple protein inactivation.

The molecular consequences include:

• Hyperstabilization of platelet pseudopods that mimics the effects of PP2A inhibition

• Significant actin polymerization defects

• Abnormal hyperphosphorylation of multiple components of the actin cytoskeleton

• Enhanced binding to fibrinogen, suggesting hyperstabilization of fibrinogen-receptor complexes

These alterations appear to be mediated through dysregulated activity of several kinases, as inhibiting upstream kinases such as PKA, PKC, or AMPK can rescue the phenotype both in vitro and in vivo . This reveals an unexpected role for MASTL in regulating actin cytoskeletal dynamics in postmitotic cells, extending its functional relevance beyond mitotic regulation. The complex molecular consequences of this mutation highlight the importance of studying MASTL in diverse cellular contexts beyond cell division .

What is the relationship between MASTL and the DNA damage response pathway?

Recent research has uncovered a significant role for MASTL in the DNA damage response (DDR) pathway, revealing a complex regulatory network. Upon DNA damage, MASTL protein levels are upregulated through a specific mechanism involving the ATM-E6AP axis . Specifically, ATM (ataxia-telangiectasia mutated) kinase phosphorylates E6AP (E3 ubiquitin-protein ligase) at Ser-218 in response to DNA damage, which inhibits E6AP-mediated ubiquitination and subsequent degradation of MASTL .

This stabilization of MASTL is functionally significant as it promotes cell cycle checkpoint recovery after DNA damage. Mechanistically, MASTL likely facilitates this recovery by inhibiting PP2A phosphatase activity, which would otherwise dephosphorylate and inactivate key mitotic regulators . This prevents premature mitotic entry until DNA damage is adequately repaired.

The MASTL-DDR relationship is bidirectional - while DNA damage regulates MASTL stability, MASTL itself influences DDR pathway components. Research suggests that MASTL modulates the phosphorylation status of DDR proteins including γ-H2AX, phospho-SMC1, phospho-CHK1, and phospho-CHK2 . This creates a regulatory feedback loop that finely tunes cellular responses to DNA damage.

For researchers investigating this pathway, it's important to note that detecting these interactions requires specific experimental approaches. Phospho-specific antibodies against E6AP Ser-218 have been developed to track this activation , and the relationship can be studied using DNA-damaging agents like etoposide or irradiation, followed by assessment of MASTL stability, ubiquitination status, and cell cycle progression markers .

What are the recommended protocols for MASTL immunoprecipitation?

For successful MASTL immunoprecipitation (IP), careful attention to protocol details is essential. Based on published methodologies, the following optimized protocol is recommended:

Lysis Buffer Composition:

• 50 mM HEPES (pH 7.5)

• 150 mM NaCl

• 1 mM DTT

• 0.5% Tween 20 (for standard IP) or

• ELB lysis buffer alternative: 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40

Critical Protocol Steps:

• Cell Preparation: Harvest cells at approximately 80% confluence and wash with cold PBS.

• Lysis: Add cold lysis buffer supplemented with both phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) and protease inhibitors (e.g., PMSF, aprotinin, leupeptin). Incubate with rotatory agitation for 30 minutes at 4°C.

• Lysate Clearing: Centrifuge at 13,000 rpm for 10 minutes at 4°C and collect the supernatant.

• Protein Quantification: Determine protein concentration using BCA assay.

• Antibody-Bead Preparation: Conjugate MASTL antibody to Protein A or G magnetic beads (2 μg antibody per 1 mg of total protein) . For reproducible results, crosslink the antibody to the beads using a suitable crosslinker.

• Immunoprecipitation: Incubate the prepared beads with lysate for 16 hours at 4°C with rotatory agitation.

• Washing: Perform at least 3-5 washes with lysis buffer.

• Elution: For Western blotting analysis, add sample buffer (containing SDS and DTT), boil for 5 minutes, and proceed with electrophoresis.

For detecting MASTL-interacting proteins or specific post-translational modifications, published studies have successfully used both monoclonal antibodies (such as the mouse monoclonal MASTL 4F9 clone) and polyclonal antibodies raised against specific regions of MASTL . The choice between these depends on the specific research question, with monoclonals often providing higher specificity for single epitopes and polyclonals offering broader detection of multiple epitopes.

How should MASTL antibodies be optimized for immunofluorescence applications?

Optimizing MASTL antibodies for immunofluorescence (IF) applications requires specific methodological considerations to achieve high signal-to-noise ratio and accurate subcellular localization. Based on published protocols, researchers should follow these optimization steps:

Sample Preparation:

• Grow cells on microscope cover glasses to appropriate confluence (70-80%).

• Fix samples using 3% formaldehyde containing 0.1% Triton X-100, which maintains cellular architecture while allowing antibody penetration .

• Permeabilize with 0.05% saponin, which creates smaller pores than Triton X-100 and better preserves cellular structures .

Blocking and Antibody Incubation:

• Block with 5% goat serum (or serum from the species of secondary antibody origin) for at least 30 minutes.

• Dilute primary MASTL antibody in blocking buffer at 1:100 to 1:500 dilution (requires optimization).

• Incubate with primary antibody for 2 hours at room temperature or overnight at 4°C .

• Use Alexa Fluor secondary antibodies at 1:2000 dilution for optimal signal with minimal background .

Critical Optimization Parameters:

• Titrate primary antibody concentration to determine optimal signal-to-noise ratio

• Compare different fixation methods (paraformaldehyde alone vs. formaldehyde with Triton X-100)

• Test various antigen retrieval methods if working with tissue sections

• Include MASTL-depleted controls (siRNA or shRNA treated cells) to confirm specificity

• Include nuclear counterstain (DAPI) to correlate MASTL localization with cell cycle phase

When imaging, use a high-quality fluorescence microscope with appropriate filter sets. Published studies have successfully employed systems such as the Zeiss Axiovert 200M inverted fluorescence microscope . For co-localization studies, use sequential scanning to prevent bleed-through between channels, particularly important when studying MASTL's relationships with other cellular structures during mitosis or following DNA damage .

What are the best practices for measuring MASTL kinase activity in vitro?

Measuring MASTL kinase activity in vitro requires carefully optimized assay conditions to obtain reliable and reproducible results. Based on published methodologies, the following best practices are recommended:

Standard In Vitro Kinase Assay Protocol:

• Reaction Components:

  • Kinase buffer: 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM DTT

  • ATP: 100 μM ATP supplemented with γ-³²P-ATP (3000 Ci/mmol) for radioactive detection

  • MASTL protein: 0.25-0.5 μM purified MASTL (recombinant or immunoprecipitated)

  • Substrate: 50 μM of either validated substrate (Arpp19) or model substrate (MBP)

• Assay Conditions:

  • Incubation time: 20-30 minutes at 30°C

  • Reaction termination: Addition of LDS Sample Buffer

  • Analysis: SDS-PAGE followed by autoradiography or phospho-specific Western blotting

• Quantification:

  • Measure radioactive signal by densitometry analysis of autoradiograms

  • Normalize activity to MASTL protein amount in each sample

  • Express as percentage of phosphorylation compared to full-length MASTL

For kinetic characterization studies, maintain constant MASTL concentration (0.25 or 0.5 μM) while varying substrate concentration (1-240 μM) . Ensure that time courses remain in the linear range for accurate initial velocity measurements.

Several validated substrates can be used for MASTL activity assays, with recombinant human Arpp19 being the most physiologically relevant . For researchers investigating novel MASTL functions, mass spectrometry-identified targets may be incorporated as substrates to explore pathway-specific activities .

How do MASTL antibodies perform in different cancer tissue types?

MASTL antibodies show variable performance across cancer tissue types, with effectiveness dependent on both antibody characteristics and tissue-specific MASTL expression patterns. Extensive immunohistochemistry (IHC) studies across cancer cohorts have provided valuable insights into optimizing detection protocols and interpreting results.

In breast cancer tissues, MASTL antibodies perform particularly well for stratifying tumor subtypes. IHC analysis of 851 breast cancer patients in the FinHer trial demonstrated that MASTL expression correlated significantly with specific cancer characteristics . The following table summarizes these correlations:

These findings indicate that MASTL antibodies can serve as effective biomarkers, particularly for identifying aggressive breast cancer subtypes . The specificity of staining in these studies was validated using MASTL-silenced controls, confirming that the observed patterns represent genuine MASTL expression rather than non-specific binding .

For researchers working with various cancer tissues, it's important to note that fixation protocols significantly impact antibody performance. Standard formalin-fixed paraffin-embedded (FFPE) tissues require antigen retrieval steps, typically heat-induced epitope retrieval in citrate buffer (pH 6.0) . Additionally, amplification systems may be necessary for detecting lower MASTL expression levels in certain tissue types. Background staining can be minimized through careful titration of primary antibody concentration and optimization of blocking conditions using normal serum from the secondary antibody species .