MAD1L1 Antibody

What is MAD1L1 and why is it significant in cell biology research?

MAD1L1 is a component of the mitotic spindle-assembly checkpoint that prevents the onset of anaphase until all chromosomes are properly aligned at the metaphase plate. It functions by recruiting MAD2 to unattached kinetochores, promoting the binding of MAD2 to CDC20, the activator for the anaphase-promoting complex . MAD1L1 has been implicated in both tumor-suppressing functions and, paradoxically, oncogenic roles in certain contexts, such as small-cell lung cancer where the MAD1L1 gene shows frequent copy number gains . Its critical role in preventing chromosomal instability makes it an important target for understanding fundamental cell cycle regulation and pathological states characterized by aneuploidy.

What are the most validated applications for MAD1L1 antibodies in research?

MAD1L1 antibodies have been validated for several research applications, with Western blot being the most commonly reported. In Western blot applications, MAD1L1 typically appears as a band at approximately 90 kDa . Other validated applications include:

• Immunocytochemistry/Immunofluorescence (ICC/IF) for localization studies

• ELISA for quantitative detection

• Immunoprecipitation for protein-protein interaction studies

When using these antibodies, researchers should note that detection has been successfully demonstrated in HeLa human cervical epithelial carcinoma cell line and Jurkat human acute T cell leukemia cell line lysates . For optimal results, each laboratory should determine appropriate dilutions for their specific application.

What are the recommended storage and handling conditions for MAD1L1 antibodies?

For optimal preservation of antibody activity, follow these evidence-based storage and handling guidelines:

• Store at -20°C to -70°C for long-term storage (up to 12 months from receipt)

• For shorter periods (up to 1 month), store at 2-8°C under sterile conditions after reconstitution

• For medium-term storage (up to 6 months), maintain at -20 to -70°C under sterile conditions after reconstitution

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles as these can significantly degrade antibody performance

• Prepare small aliquots upon initial thawing to minimize freeze-thaw cycles

How should I optimize Western blot protocols for MAD1L1 detection?

Optimization of Western blot protocols for MAD1L1 detection requires attention to several parameters:

• Sample preparation: Use appropriate lysis buffers that preserve protein integrity while efficiently extracting nuclear proteins.

• Antibody dilution: Start with manufacturer-recommended dilutions (typically 1/1000 or 1 μg/mL ) and adjust as needed.

• Detection system: For polyclonal antibodies raised in rabbit, an HRP-conjugated anti-rabbit IgG secondary antibody is appropriate. For goat-derived antibodies, use HRP-conjugated Anti-Goat IgG Secondary Antibody (e.g., Catalog # HAF017) .

• Membrane type: PVDF membranes have been successfully used for MAD1L1 detection .

• Blocking conditions: Optimize blocking buffer composition to minimize background while preserving specific signal.

• Controls: Include positive controls such as HeLa or Jurkat cell lysates where MAD1L1 expression has been confirmed .

How can I use MAD1L1 antibodies to investigate the spindle assembly checkpoint in cancer models?

Investigation of SAC function in cancer models using MAD1L1 antibodies requires multi-faceted approaches:

• Immunolocalization studies: Use immunofluorescence with MAD1L1 antibodies to visualize protein localization at kinetochores during mitosis. Proper SAC function shows MAD1L1 localization at unattached kinetochores during prometaphase, with signal disappearing upon attachment.

• Protein-protein interaction analysis: Use co-immunoprecipitation with MAD1L1 antibodies to assess interactions with other SAC components such as MAD2 and checkpoint activation.

• Expression correlation with aneuploid phenotypes: Quantify MAD1L1 protein levels via Western blot and correlate with cellular aneuploidy measured by flow cytometry or karyotyping. Functional studies have demonstrated that biallelic mutations in MAD1L1 resulted in lack of full-length protein and deficient SAC response, resulting in approximately 30-40% of aneuploid blood cells .

• Mitotic timing assessment: Combine MAD1L1 immunostaining with live-cell imaging to evaluate mitotic duration and checkpoint robustness in response to spindle poisons.

What are the considerations when using antibodies to detect the RARS-MAD1L1 fusion protein?

Detection of the RARS-MAD1L1 fusion protein presents unique challenges requiring specific considerations:

• Antibody selection: Use antibodies targeting the C-terminal region of MAD1L1 (e.g., catalog no. A300-355A) or the N-terminal region of RARS (e.g., Novus Biologicals, catalog no. PAB28524) depending on whether you want to detect the fusion or differentiate it from wild-type proteins.

• Fusion verification strategy: Employ a dual-detection approach using antibodies against both RARS and MAD1L1 to confirm co-localization or co-immunoprecipitation.

• Specificity controls: Include samples known to express the fusion (e.g., C666-1 cells) and those that don't as controls. The fusion gene has been found in 10.03% (35/349) of primary nasopharyngeal carcinoma biopsies and 10.7% (9/84) of head and neck cancer samples .

• Detection sensitivity considerations: Western blot may require longer exposure times or more sensitive detection systems due to potentially lower expression levels of the fusion protein compared to wild-type proteins.

• Functional validation: Complement antibody detection with functional assays, as RARS-MAD1L1 has been shown to increase cell proliferation, colony formation, and tumorigenicity in vitro .

How can I assess MAD1L1 expression in relation to chromosomal instability syndromes?

To investigate MAD1L1's role in chromosomal instability syndromes:

• Quantitative expression analysis: Use Western blot with MAD1L1 antibodies to quantify protein levels in patient-derived cells compared to healthy controls.

• Functional checkpoint assay: Assess SAC activity in patient cells using nocodazole treatment followed by flow cytometry for mitotic index determination, complemented with MAD1L1 immunostaining.

• Aneuploidy correlation: Combine MAD1L1 expression analysis with single-cell DNA content analysis or karyotyping to establish correlations with aneuploidy levels. Research has shown that biallelic germline mutations in MAD1L1 can induce a syndrome of aneuploidy with high tumor susceptibility .

• Downstream pathway analysis: Investigate consequences of MAD1L1 dysfunction using antibodies against markers of mitochondrial stress and inflammation, as single-cell RNA analysis has identified mitochondrial stress accompanied by systemic inflammation with enhanced interferon and NFκB signaling in both aneuploid and euploid cells from affected patients .

What troubleshooting steps should I take when experiencing non-specific binding with MAD1L1 antibodies?

When encountering non-specific binding issues:

• Antibody validation: Verify antibody specificity using knockout/knockdown controls. Published studies have used wild type S. cerevisiae whole cell lysate compared with mad1 knockout S. cerevisiae lysate .

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Increase blocking time and/or concentration

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions

• Titration experiments: Perform a dilution series to determine optimal antibody concentration that maximizes specific signal while minimizing background.

• Pre-absorption controls: Pre-incubate antibody with excess antigen peptide to confirm specificity of bands/signals.

• Alternative antibody selection: Consider antibodies targeting different epitopes. For example, choose between antibodies against the N-terminal region (Met1-Asp350) versus C-terminal region of MAD1L1.

How can I integrate MAD1L1 protein studies with epigenetic research on psychiatric disorders?

Emerging research has linked MAD1L1 methylation with psychiatric phenotypes . To integrate protein studies with epigenetic research:

• Correlation analysis: Combine Western blot quantification of MAD1L1 protein levels with methylation analysis at specific CpG sites (particularly cg02825527, cg19624444, and cg18302629) that have shown associations with psychiatric phenotypes .

• Tissue-specific considerations: Remember that the direction of associations between psychiatric phenotypes and methylation appears to differ between whole blood and brain samples for certain CpG sites (cg02825527 and cg19624444) .

• Expression-methylation relationship: Investigate the relationship between methylation at cg18302629 and cg19624444 and MAD1L1 transcript levels, as these have shown potential links to MAD1L1 expression in CD14+ cells .

• Cell type-specific analysis: Consider examining MAD1L1 protein expression in specific immune cell populations relevant to psychiatric disorders, particularly given the evidence of clonal expansions of γδ T cells with chromosome 18 gains and enhanced cytotoxic profiles in individuals with MAD1L1 mutations .

What emerging research areas might benefit from MAD1L1 antibody applications?

Several cutting-edge research areas could benefit from MAD1L1 antibody applications:

• Cancer stem cell biology: Given the role of RARS-MAD1L1 in inducing cancer stem cell-like properties and chemo/radio-resistance , MAD1L1 antibodies could be valuable in studying cancer stem cell populations.

• Immunological consequences of aneuploidy: Research into how MAD1L1 dysfunction affects immune system function, particularly in light of findings showing clonal expansions of specific immune cell populations with chromosomal gains in individuals with MAD1L1 mutations .

• Psychiatric genomics: Further exploration of the relationship between MAD1L1 protein expression, methylation status, and psychiatric phenotypes .

• Therapeutic targeting: Development of approaches to modulate MAD1L1 function or downstream pathways for potential therapeutic benefit in cancers characterized by SAC dysregulation.

• Developmental biology: Investigation of MAD1L1's role in embryonic development, given that lack of Mad1l1 is lethal in mice during early development .