MASTL Antibody, HRP conjugated

What is MASTL and why is it significant for cell cycle research?

MASTL (also known as Greatwall kinase or GWL) is a serine/threonine protein kinase that plays a crucial role in mitotic regulation. MASTL functions within the cell cycle by repressing the activity of mitotic protein phosphatases PP1 and PP2A during entry into mitosis, working alongside CDK1. Recent research has revealed that MASTL is frequently upregulated in several cancers and correlates with cancer progression, poor patient survival, and tumor recurrence. The protein has been identified as a potential target for therapeutic intervention as ectopic expression of MASTL in tumor cells promotes cell proliferation . Understanding MASTL function is therefore critical for both basic cell biology and translational cancer research.

How does the HRP conjugation of MASTL antibodies benefit detection methods compared to unconjugated alternatives?

HRP (horseradish peroxidase) conjugation provides direct enzymatic detection capabilities to MASTL antibodies, eliminating the need for secondary antibody incubation steps. This offers several methodological advantages:

• Reduced protocol time and complexity in ELISA and immunoblotting applications

• Decreased background signal by eliminating potential cross-reactivity from secondary antibodies

• Enhanced sensitivity through direct enzymatic signal amplification

• Improved reproducibility by reducing protocol variability

What are the optimal storage and handling conditions for maintaining MASTL antibody, HRP conjugated activity?

To maintain optimal activity of HRP-conjugated MASTL antibodies, follow these evidence-based storage and handling protocols:

Following reconstitution, HRP-conjugated MASTL antibodies remain stable for approximately 6 months at -20°C to -70°C under sterile conditions . For maximum sensitivity in applications, use freshly thawed aliquots whenever possible.

How can I optimize MASTL antibody concentration for detecting DNA damage-induced MASTL upregulation?

Recent research has revealed that DNA damage induces significant upregulation of MASTL protein levels through post-translational mechanisms . To optimize detection of this phenomenon:

• Determine baseline expression: First establish baseline MASTL expression in your specific cell type under normal conditions using a dilution series (typically 1:500 to 1:5000) of HRP-conjugated MASTL antibody.

• Damage induction protocol: Treat cells with DNA damaging agents such as doxorubicin (DOX), hydroxyurea (HU), or camptothecin (CPT) at research-established concentrations. MASTL upregulation occurs generally within hours post-treatment and coincides with ATM/ATR signaling activation .

• Antibody titration: For cells with DNA damage-induced MASTL upregulation, a higher dilution (1:2000 to 1:5000) may be appropriate to prevent signal saturation.

• Control markers: Include parallel detection of ATM/ATR substrate phosphorylation and replication protein A (RPA) phosphorylation to confirm DNA damage response activation .

• Quantification: Normalize MASTL signal to an internal loading control that remains stable during DNA damage response, such as GAPDH.

This optimization approach ensures accurate detection of the physiologically relevant upregulation of MASTL that specifically occurs during DNA damage response pathways.

What are the key considerations when using MASTL antibody, HRP conjugated in cell-based ELISA systems?

Cell-based ELISA systems using HRP-conjugated MASTL antibodies require specific technical considerations:

• Cell density optimization: The MASTL Cell-Based ELISA system is optimized for >5000 cells per well. Lower densities may yield unreliable results due to insufficient signal-to-noise ratio .

• Normalization strategies: Two complementary approaches are recommended:

  • Include anti-GAPDH antibody as an internal positive control

  • Apply Crystal Violet whole-cell staining post-HRP measurement to normalize results to cell density

• Adherent vs. suspension cells: Both cell types can be analyzed, but attachment factors may be needed for suspension cells to ensure consistent cell retention during washing steps .

• Stimulation conditions: When investigating how different conditions affect MASTL expression, maintain consistent intervals between stimulation and fixation across experimental groups.

• Signal development time: Optimal development time for the colorimetric reaction is approximately 30 minutes, but should be empirically determined for each cell type and condition.

The complete assay typically requires 4.5 hours and provides qualitative determination of MASTL concentration through an indirect ELISA format where MASTL is captured by specific antibodies and then detected via HRP-conjugated secondary antibodies .

How can I distinguish between phosphorylated and non-phosphorylated forms of MASTL when using HRP-conjugated antibodies?

Distinguishing phosphorylation states of MASTL is crucial since its activity is regulated by multiple phosphorylation events. Consider these methodological approaches:

• Phosphatase treatment controls: Split your samples and treat one set with lambda phosphatase before immunodetection to establish mobility shifts associated with phosphorylation.

• Complementary antibodies: Use phospho-specific MASTL antibodies alongside total MASTL HRP-conjugated antibodies in parallel samples.

• SDS-PAGE optimization: Use Phos-tag™ acrylamide gels or lower percentage gels (6-8%) to better resolve phosphorylated MASTL species, which typically migrate with reduced mobility.

• Combining with immunoprecipitation: For more sensitive analysis, immunoprecipitate MASTL first, then analyze with phospho-specific antibodies or mass spectrometry.

• Correlation with cell cycle phase: MASTL phosphorylation state correlates with cell cycle progression, particularly during mitotic entry when CDK1-mediated phosphorylation occurs.

During mitotic exit, dephosphorylation of MASTL occurs in a sequential manner, first by PP1 and then by PP2A, which can be tracked using these techniques to understand the temporal dynamics of MASTL regulation .

How can I validate the specificity of MASTL antibody, HRP conjugated in my experimental system?

Validating antibody specificity is essential for generating reliable research data. For HRP-conjugated MASTL antibodies, implement these validation strategies:

• Genetic controls: Use CRISPR-Cas9-mediated MASTL knockout or siRNA-mediated knockdown cells as negative controls to confirm signal specificity .

• Overexpression controls: Compare signal in cells with endogenous MASTL expression versus those overexpressing recombinant MASTL.

• Peptide competition: Pre-incubate the antibody with excess immunogen peptide (recombinant Human Serine/threonine-protein kinase greatwall protein, specifically amino acids 612-740) to block specific binding sites .

• Cross-species reactivity assessment: MASTL antibodies may exhibit different specificity across species. Confirm reactivity patterns match the predicted evolutionary conservation pattern.

• Subcellular localization: Verify that the detected signal matches the expected cytoplasmic localization pattern for MASTL as shown in validated immunofluorescence studies .

A comprehensive validation approach increases confidence in experimental results and ensures that observed signals genuinely represent MASTL protein rather than non-specific interactions or artifacts.

What are the known cross-reactivity concerns with MASTL antibodies and how can they be addressed?

While MASTL antibodies are designed for specificity, potential cross-reactivity with related kinases or proteins can occur. Consider these methodological approaches to address cross-reactivity concerns:

• Sequence homology analysis: MASTL belongs to the AGC kinase family with sequence similarities to other members. Verify that your antibody was raised against unique MASTL sequences (such as amino acids 612-740 or 39-158) rather than highly conserved kinase domains .

• Western blot profile analysis: MASTL has a molecular weight of approximately 97 kDa. Confirm that your HRP-conjugated antibody detects a predominant band at this size.

• Multiple antibody approach: Use antibodies targeting different epitopes of MASTL to confirm findings.

• Immunodepletion experiments: Deplete MASTL from samples using one antibody, then test for remaining signal using the HRP-conjugated antibody.

• Mass spectrometry validation: For definitive validation, immunoprecipitate proteins detected by the antibody and analyze by mass spectrometry to confirm MASTL identity.

Known potential cross-reactive proteins include other members of the microtubule-associated serine/threonine kinase family due to sequence similarities in functional domains, though properly validated antibodies specifically targeting amino acids 612-740 of human MASTL show high specificity .

How can MASTL antibody, HRP conjugated be utilized to study the ATM-E6AP-MASTL axis in DNA damage response?

Recent research has uncovered a critical ATM-E6AP-MASTL regulatory axis in DNA damage response . To investigate this pathway using HRP-conjugated MASTL antibodies:

• Temporal analysis: Track MASTL protein levels at defined time points (0, 2, 4, 8, 12, 24 hours) following DNA damage induction with doxorubicin, hydroxyurea, or camptothecin.

• ATM/ATR inhibition studies: Co-treat cells with DNA damaging agents and ATM/ATR inhibitors (such as caffeine) to demonstrate the dependence of MASTL upregulation on ATM/ATR signaling .

• E6AP modulation: Combine MASTL detection with E6AP knockdown or overexpression to investigate the inverse relationship between E6AP levels and MASTL stability.

• Protein stability assessment: Perform cycloheximide chase experiments with and without DNA damage to measure MASTL protein half-life changes.

• Ubiquitination analysis: Couple immunoprecipitation with ubiquitin detection to assess how DNA damage affects MASTL ubiquitination status.

This approach allows researchers to dissect the mechanism by which ATM/ATR activation inhibits E6AP-mediated MASTL degradation, resulting in MASTL protein accumulation during DNA damage response .

What methods can improve the detection sensitivity of low-abundance MASTL in primary tissue samples?

Primary tissue samples often contain lower abundance of target proteins compared to cell lines, requiring modified approaches for effective MASTL detection:

• Signal amplification systems: Use tyramide signal amplification (TSA) with HRP-conjugated MASTL antibodies to enhance sensitivity by approximately 10-100 fold.

• Sample preparation optimization:

  • Use optimized extraction buffers containing protease and phosphatase inhibitors

  • Include 50% glycerol in sample preparation to stabilize MASTL protein

  • Consider tissue-specific extraction protocols to maximize protein recovery

• Concentration techniques:

  • Immunoprecipitate MASTL before detection to concentrate the target

  • Use larger amounts of starting material (50-100 μg total protein versus standard 20-30 μg)

• Detection system modifications:

  • Utilize enhanced chemiluminescent substrates specifically designed for low-abundance proteins

  • Extend primary antibody incubation time to 24-48 hours at 4°C

• Automated imaging analysis: Apply computational image analysis to quantify signals at the lower detection limit, using appropriate statistical methods to distinguish signal from background.

For tissue microarrays or histological sections, the optimal concentration of HRP-conjugated MASTL antibody is typically 10-20 μg/mL with extended incubation times compared to standard protocols used for cultured cells .

How should experimental protocols be modified when investigating MASTL in different cancer cell lines?

Cancer cell lines exhibit varied MASTL expression levels and regulation patterns, requiring protocol adaptations:

• Baseline expression determination: Before comparative studies, quantify the basal MASTL expression levels across your panel of cancer cell lines using standardized western blotting with recombinant MASTL protein standards.

• Cell line-specific optimization:

  • HeLa cells show detectable MASTL expression that responds to DNA damage with clear upregulation

  • SCC38 cells demonstrate robust MASTL protein stability changes in response to hydroxyurea and ionizing radiation

  • HEK293 cells exhibit MASTL upregulation within hours of doxorubicin treatment

• Cell cycle synchronization: Since MASTL expression and phosphorylation status varies throughout the cell cycle, synchronize cells before treatment to reduce variability.

• Detection sensitivity adjustment: For cell lines with lower MASTL expression, reduce the antibody dilution factor (1:500 instead of 1:1000) and extend incubation times.

• Cancer-specific pathway analysis: When studying MASTL in cancer contexts, include parallel detection of cancer-relevant pathways like DNA damage response markers or cell cycle regulators to contextualize MASTL data.

The consistent finding across multiple cancer cell lines is that DNA damage induces MASTL protein upregulation through post-translational mechanisms, while other mitotic kinases (CDK1/cyclin B, Aurora A, Aurora B, PLK1) do not show similar upregulation patterns .

What are the recommended controls when studying MASTL protein stability using HRP-conjugated antibodies?

When investigating MASTL protein stability with HRP-conjugated antibodies, incorporate these essential controls:

• Protein synthesis inhibition control: Use cycloheximide (CHX) to block new protein synthesis and accurately measure degradation rates of existing MASTL protein .

• Proteasome inhibition: Include MG132 treatment to confirm the proteasome-dependent nature of MASTL degradation.

• Half-life determination standards: Generate a degradation curve with at least 5 time points (0, 2, 4, 8, 12 hours) following cycloheximide treatment to calculate accurate half-life values .

• Reference proteins: Include parallel detection of proteins with well-established half-lives:

  • Short-lived control: c-Myc (t½ ≈ 20-30 minutes)

  • Medium-lived control: p53 (t½ ≈ 5-20 minutes under normal conditions)

  • Long-lived control: α-tubulin (t½ > 24 hours)

• E6AP modulation: Since E6AP has been identified as a ubiquitin ligase for MASTL, include E6AP-depleted conditions as a positive control for extended MASTL stability .

Studies have shown that DNA damage treatment (such as with CPT or HU) significantly increases MASTL protein stability, prolonging its half-life in multiple cell lines including HeLa, SCC38, and HEK293 .

How can researchers distinguish between the effects of DNA damage on MASTL expression versus activation?

DNA damage induces both MASTL upregulation and activation, which must be distinguished methodologically:

• Protein level versus activity detection:

  • Use HRP-conjugated MASTL antibodies to quantify total protein levels

  • Employ kinase activity assays with recombinant substrates to directly measure MASTL catalytic activity

• Phosphorylation site mapping:

  • Monitor specific activating phosphorylation sites (such as CDK1-mediated phosphorylation)

  • Track inhibitory phosphorylation states that regulate MASTL activity

• Downstream substrate analysis:

  • Measure phosphorylation of MASTL substrates (such as ENSA and ARPP19)

  • Quantify PP2A activity as an inverse indicator of MASTL function

• Temporal resolution studies:

  • Perform fine-grained time course experiments to distinguish rapid activation (minutes) from slower protein accumulation (hours)

  • Use synchronized cell populations to control for cell cycle-dependent variations

• Genetic complementation:

  • Express catalytically inactive MASTL mutants to separate protein presence from activity

  • Use phosphomimetic or phospho-dead MASTL mutants to assess the role of specific modifications

Research has demonstrated that while MASTL protein upregulation occurs following DNA damage through ATM/ATR-dependent inhibition of E6AP-mediated degradation, this distinct from the rapid activation of MASTL via phosphorylation that occurs during normal mitotic entry .