Phospho-MAD1L1 (S428) Antibody

What is Phospho-MAD1L1 (S428) Antibody and what cellular processes does it help investigate?

Phospho-MAD1L1 (S428) Antibody specifically recognizes the MAD1L1 protein only when phosphorylated at the serine 428 residue. This antibody serves as a crucial tool for investigating mitotic spindle assembly checkpoint mechanisms, which prevent the onset of anaphase until all chromosomes are properly aligned at the metaphase plate . MAD1L1 (also known as MAD1, TXBP181) functions as a component of this checkpoint system, forming a heterotetrameric complex with MAD2L1 at unattached kinetochores during prometaphase . The phosphorylation at S428 represents a specific post-translational modification that regulates MAD1L1 function in this critical cell cycle control process .

What applications is Phospho-MAD1L1 (S428) Antibody validated for?

Based on comprehensive validation studies, Phospho-MAD1L1 (S428) Antibody has been specifically tested and confirmed effective in the following applications:

It should be noted that while these applications have been validated by manufacturers, researchers may need to optimize conditions for their specific experimental systems .

What is the recommended storage protocol for maintaining antibody stability?

To maintain optimal antibody activity, Phospho-MAD1L1 (S428) Antibody should be stored at -20°C or -80°C upon receipt . For short-term storage and frequent use, the antibody can be kept at 4°C for up to one month . The antibody is typically supplied in liquid form in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide as a preservative . It is critical to avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of binding activity . Aliquoting the antibody upon first thaw is recommended for laboratories that will use it intermittently over extended periods .

How should researchers optimize Phospho-MAD1L1 (S428) Antibody dilutions for immunohistochemistry?

For optimal immunohistochemistry results with Phospho-MAD1L1 (S428) Antibody, a methodical approach to dilution optimization is recommended:

• Begin with the manufacturer's suggested range of 1:100-1:300 .

• Perform a dilution series experiment using positive control tissue known to express phosphorylated MAD1L1.

• Include appropriate negative controls, such as:

  • Omission of primary antibody

  • Blocking with the phosphopeptide immunogen

  • Use of tissue from MAD1L1 knockout models if available

The optimal dilution should provide clear specific staining with minimal background. When using paraffin-embedded tissues, ensure proper antigen retrieval, as phospho-epitopes can be particularly sensitive to fixation conditions . Blocking with the phosphopeptide can confirm signal specificity, as demonstrated in validation studies showing complete signal abolishment when the antibody is pre-incubated with the phosphopeptide .

What controls should be included when using Phospho-MAD1L1 (S428) Antibody in cell cycle research?

When investigating MAD1L1 phosphorylation in cell cycle research, comprehensive controls are essential:

• Cell cycle synchronization controls:

  • Include samples from cells synchronized at different cell cycle stages (G1, S, G2, prometaphase, metaphase, anaphase)

  • MAD1L1 becomes hyperphosphorylated in late S through M phases

• Treatment controls:

  • Mitotic spindle damage positive control (e.g., nocodazole-treated cells)

  • Phosphatase inhibitor controls to preserve phosphorylation status

  • Phosphatase-treated negative controls to remove phosphorylation

• Localization controls:

  • Co-staining with kinetochore markers during prometaphase when MAD1L1 localizes to kinetochores

  • Co-staining with TPR to verify co-localization at the nuclear envelope

• Specificity controls:

  • Blocking with the phospho-specific peptide used as immunogen

  • Non-phosphorylated peptide control to confirm phospho-specificity

These controls will help distinguish specific phosphorylation-dependent signals from background and confirm the biological relevance of observations in cell cycle studies.

How can researchers address weak or absent signals when using Phospho-MAD1L1 (S428) Antibody?

When encountering weak or absent signals with Phospho-MAD1L1 (S428) Antibody, consider the following methodological approach:

• Verify phosphorylation status preservation:

  • Include phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride) in all buffers

  • Minimize sample handling time before fixation

  • Use phospho-protein preservation fixatives

• Optimize antigen retrieval for phospho-epitopes:

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize buffer pH (typically pH 9.0 works better for phospho-epitopes)

  • Extend retrieval time incrementally

• Signal amplification strategies:

  • Employ tyramide signal amplification systems

  • Use biotin-streptavidin amplification (if background permits)

  • Consider longer primary antibody incubation at 4°C (overnight)

• Verify with positive control tissues:

  • Use tissues with known high levels of phosphorylated MAD1L1

  • Include mitotic cell-enriched samples (e.g., rapidly dividing tissues)

• Antibody quality check:

  • Verify antibody hasn't undergone multiple freeze-thaw cycles

  • Check storage conditions and expiration date

  • Consider testing a new lot if problems persist

Remember that phosphorylation is often transient and can be lost during sample preparation, so careful attention to preservation techniques is crucial for detecting this post-translational modification .

What strategies can reduce non-specific background when using Phospho-MAD1L1 (S428) Antibody?

To minimize non-specific background when working with Phospho-MAD1L1 (S428) Antibody, implement these evidence-based strategies:

• Optimize blocking conditions:

  • Extend blocking time to 1-2 hours at room temperature

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Consider adding 0.1-0.3% Triton X-100 to blocking buffer for better penetration

• Antibody dilution optimization:

  • Perform careful titration experiments beyond manufacturer recommendations

  • Prepare antibody dilutions in fresh buffer containing blocking agent

  • Pre-absorb antibody with non-specific proteins if cross-reactivity is suspected

• Washing protocol enhancement:

  • Increase washing duration and number of washes (6-8 washes of 5-10 minutes each)

  • Use gentle agitation during washing steps

  • Add 0.05-0.1% Tween-20 to wash buffers

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Dilute secondary antibody more than typically recommended

  • Include negative controls omitting primary antibody to identify secondary antibody background

• Sample-specific techniques:

  • For tissues with high endogenous peroxidase activity, use additional quenching steps

  • For tissues with high biotin content, use biotin-free detection systems

  • Block endogenous Fc receptors with appropriate blocking reagents

These approaches should systematically reduce background while preserving specific signal for phosphorylated MAD1L1 .

How can Phospho-MAD1L1 (S428) Antibody be used to investigate spindle assembly checkpoint dysfunction in cancer?

Phospho-MAD1L1 (S428) Antibody offers powerful insights into spindle assembly checkpoint (SAC) dysfunction in cancer through multiple experimental approaches:

• Comparative phosphorylation analysis:

  • Quantify phospho-MAD1L1 levels in matched tumor/normal tissue pairs using immunohistochemistry

  • Correlate phosphorylation levels with chromosomal instability markers

  • Examine relationship between phospho-MAD1L1 and clinical outcomes

• Subcellular localization studies:

  • Use immunofluorescence to track phospho-MAD1L1 localization during mitosis in cancer cells

  • Compare kinetochore recruitment dynamics between normal and cancer cells

  • Investigate co-localization with other SAC components (MAD2, BUB1, etc.)

• Checkpoint signaling analysis:

  • Examine how cancer-associated mutations affect MAD1L1 phosphorylation at S428

  • Investigate kinase-phosphatase balance regulating this modification in cancer cells

  • Study the impact of cancer therapeutic agents on MAD1L1 phosphorylation

• Isoform-specific effects:

  • Determine how MAD1L1 isoform 3, which sequesters MAD2L1 in the cytoplasm causing chromosomal instability in hepatocellular carcinomas, affects S428 phosphorylation

  • Compare phosphorylation patterns across cancer subtypes expressing different MAD1L1 isoforms

• Mechanistic studies:

  • Investigate the interplay between BUB1 and TTK (known to phosphorylate MAD1L1) in cancer cells

  • Examine how altered S428 phosphorylation affects MAD1-MAD2 complex formation

These approaches leverage the specificity of Phospho-MAD1L1 (S428) Antibody to provide mechanistic insights into how SAC dysfunction contributes to genomic instability in cancer .

What methodological approaches allow for quantitative assessment of MAD1L1 phosphorylation dynamics during cell cycle progression?

To quantitatively assess MAD1L1 phosphorylation dynamics throughout the cell cycle, several sophisticated methodological approaches can be employed:

• Time-resolved immunofluorescence microscopy:

  • Synchronize cells and collect samples at defined time points

  • Use Phospho-MAD1L1 (S428) Antibody in immunofluorescence (dilution 1:50-1:200)

  • Employ automated image analysis to quantify signal intensity changes

  • Co-stain with cell cycle phase markers for precise temporal correlation

• Flow cytometry-based analysis:

  • Perform multiparameter flow cytometry combining:

    • DNA content staining (for cell cycle phase identification)

    • Phospho-MAD1L1 (S428) antibody detection

    • Mitotic markers (e.g., phospho-histone H3)

  • Create bivariate plots to track phosphorylation across cell cycle phases

• Quantitative phosphoproteomics integration:

  • Combine immunoprecipitation using Phospho-MAD1L1 (S428) Antibody with mass spectrometry

  • Employ SILAC or TMT labeling for precise quantification across cell cycle stages

  • Map S428 phosphorylation in the context of the full MAD1L1 phosphorylation profile

• Live-cell imaging approaches:

  • Generate cell lines expressing fluorescently-tagged MAD1L1

  • Develop phospho-specific biosensors for S428

  • Track phosphorylation dynamics in real-time during mitotic progression

• Phospho-ELISA quantification:

  • Develop quantitative ELISA using Phospho-MAD1L1 (S428) Antibody (dilution 1:5000)

  • Generate standard curves with known quantities of phosphorylated peptide

  • Compare phosphorylation levels across synchronized cell populations

These approaches collectively provide comprehensive quantitative assessment of MAD1L1 phosphorylation dynamics, revealing how this modification correlates with specific cell cycle transitions and checkpoints .

How does phosphorylation at S428 alter MAD1L1 function compared to other phosphorylation sites?

The phosphorylation of MAD1L1 at S428 represents a specific regulatory modification with distinct functional implications compared to other phosphorylation sites:

• Functional specificity:

  • S428 phosphorylation appears to be particularly important for MAD1L1's role in the spindle assembly checkpoint

  • Unlike some other sites, S428 phosphorylation is specifically targeted by BUB1 kinase

  • This phosphorylation event contributes to the hyperphosphorylated state of MAD1L1 observed in late S through M phases

• Structural implications:

  • S428 resides within the amino acid range 394-443 , a region important for protein-protein interactions

  • Phosphorylation at this site likely induces conformational changes that influence:

    • MAD1L1 recruitment to kinetochores

    • Interaction with binding partners, particularly MAD2L1

    • Formation of the mitotic checkpoint complex

• Comparative phosphorylation profile:

  • MAD1L1 is phosphorylated at multiple sites by different kinases:

    • BUB1 targets S428 and other sites

    • TTK (Mps1) phosphorylates distinct MAD1L1 residues

  • Each phosphorylation site contributes to a phospho-code that fine-tunes MAD1L1 function

  • S428 phosphorylation appears to be especially critical for checkpoint signaling

• Temporal dynamics:

  • Unlike constitutive phosphorylation sites, S428 phosphorylation shows cell cycle-dependent regulation

  • This site becomes increasingly phosphorylated as cells progress from late S-phase through mitosis

  • Phosphorylation is particularly elevated after mitotic spindle damage

Understanding the specific contribution of S428 phosphorylation within the broader context of MAD1L1 regulation provides crucial insights into the molecular mechanisms of cell cycle checkpoint control .

What is known about the kinases and phosphatases that regulate MAD1L1 S428 phosphorylation?

The phosphorylation state of MAD1L1 at S428 is regulated through a dynamic interplay of specific kinases and phosphatases:

• Kinases identifying MAD1L1 S428 as a substrate:

  • BUB1 kinase: Definitively identified as a kinase that phosphorylates MAD1L1 at S428

    • Functions primarily during early mitosis at kinetochores

    • Phosphorylation contributes to MAD1L1 hyperphosphorylation in late S through M phases

  • TTK (Mps1) kinase: Also contributes to MAD1L1 phosphorylation

    • Critical for spindle assembly checkpoint activation

    • May create a phosphorylation cascade affecting multiple sites including S428

• Regulatory context of phosphorylation:

  • Phosphorylation increases substantially after mitotic spindle damage

  • This modification likely occurs at unattached kinetochores during prometaphase

  • The phosphorylation event is part of a signaling network that includes:

    • BUB1-mediated phosphorylation

    • MAD2L1 recruitment and conversion from open to closed conformation

    • Mitotic checkpoint complex assembly

• Phosphatases and dephosphorylation dynamics:

  • While specific phosphatases targeting S428 are less characterized, candidates include:

    • PP1 (Protein Phosphatase 1)

    • PP2A (Protein Phosphatase 2A)

  • Dephosphorylation likely occurs during checkpoint silencing when chromosomes achieve proper attachment

  • The balance of kinase/phosphatase activity determines the phosphorylation status at different cell cycle stages

• Experimental assessment approaches:

  • Kinase inhibitor studies (BUB1 and TTK inhibitors)

  • Phosphatase inhibitor treatments

  • Kinase/phosphatase knockdown or knockout studies

  • In vitro kinase/phosphatase assays with recombinant proteins

Understanding this regulatory network provides insights into how MAD1L1 phosphorylation at S428 is dynamically controlled during normal cell cycle progression and in response to mitotic stress .

How can Phospho-MAD1L1 (S428) Antibody be combined with other molecular tools for comprehensive mitotic checkpoint analysis?

A sophisticated integrated approach combining Phospho-MAD1L1 (S428) Antibody with complementary molecular tools enables comprehensive analysis of mitotic checkpoint dynamics:

• Multiplexed immunofluorescence strategies:

  • Combine Phospho-MAD1L1 (S428) Antibody with antibodies against:

    • Total MAD1L1 (to calculate phosphorylation ratios)

    • MAD2L1 (for checkpoint complex formation)

    • Centromere/kinetochore markers (CENP proteins, Hec1)

    • BUB1 and TTK kinases (upstream regulators)

  • Use spectral unmixing microscopy for multi-protein localization analysis

  • Apply proximity ligation assays (PLA) to detect phospho-MAD1L1 interactions with binding partners

• Genetic engineering integration:

  • CRISPR/Cas9 editing to generate:

    • S428 phospho-mutant cell lines (S428A)

    • Phospho-mimetic mutants (S428D/E)

    • Fluorescently tagged MAD1L1 for live imaging

  • Compare antibody staining in wild-type vs. mutant backgrounds

  • Rescue experiments to confirm specificity

• Biochemical approach combinations:

  • Immunoprecipitation with Phospho-MAD1L1 (S428) Antibody followed by:

    • Mass spectrometry to identify interaction partners

    • Western blotting for known binding proteins

    • ChIP-seq for any chromatin associations

  • Sequential immunoprecipitations to isolate specific subcomplexes

• High-content screening applications:

  • Develop Phospho-MAD1L1 (S428) Antibody-based screens to identify:

    • Compounds affecting SAC phosphorylation

    • Genetic regulators of MAD1L1 phosphorylation

    • Cell cycle perturbations affecting S428 phosphorylation

  • Combine with automated image analysis for quantitative phenotyping

This integrated approach leverages the specificity of Phospho-MAD1L1 (S428) Antibody within a broader technological framework to provide systems-level insights into mitotic checkpoint regulation .

What are the considerations for using Phospho-MAD1L1 (S428) Antibody in different model organisms?

When extending Phospho-MAD1L1 (S428) Antibody applications across different model organisms, researchers should consider several critical factors:

• Species reactivity and sequence conservation:

  • Confirmed reactivity: Human is the primary validated species

  • Extended reactivity: Some antibody products report reactivity with rat and mouse samples

  • Sequence analysis: Researchers should perform sequence alignment of the region containing S428 (AA 394-443) across species of interest:

    Species	Sequence Conservation	Expected Reactivity
    Human	Reference sequence	Validated
    Mouse	High conservation	Likely reactive
    Rat	High conservation	Likely reactive
    Other mammals	Moderate to high	Variable
    Non-mammals	Lower conservation	Less likely

• Validation requirements for cross-species applications:

  • Western blot validation using positive control samples

  • Inclusion of phosphatase-treated negative controls

  • Peptide competition assays with species-specific phosphopeptides

  • Side-by-side comparison with species-specific antibodies if available

• Application-specific considerations:

  • IHC: Optimize antigen retrieval conditions for each species' tissue fixation methods

  • IF: Account for species differences in subcellular localization patterns

  • ELISA: Develop species-specific standard curves with appropriate peptides

• Biological context variations:

  • Cell cycle timing differences between species

  • Checkpoint signaling variations across evolutionary distance

  • Expression level differences requiring antibody dilution adjustments

  • Potential phosphorylation site variations affecting epitope recognition

• Controls for evolutionary divergence:

  • Include wild-type versus MAD1L1 knockout/knockdown samples

  • Use phospho-mimetic and non-phosphorylatable mutants as controls

  • Consider developing species-specific phospho-antibodies for highly divergent organisms