MAD2L1BP Antibody

What is MAD2L1BP and what is its biological function?

MAD2L1BP (MAD2L1 Binding Protein), also known as p31comet, functions primarily to silence the spindle checkpoint, allowing mitosis to proceed through anaphase. It achieves this by binding to MAD2L1 after it dissociates from the MAD2L1-CDC20 complex. MAD2L1BP plays an active role in removing MAD2 from p55CDC and tethering MAD2 for checkpoint silencing, thereby coordinating cell cycle events in late mitosis . The protein contains highly conserved central 'α-helix'- and flanked 'β-sheet'-organized domains that structurally mimic MAD2 and interact at the MAD2 dimerization interface . This interaction is essential for its role as an adaptor that binds MAD2 and recruits TRIP13.

What are the validated applications for MAD2L1BP antibodies?

MAD2L1BP antibodies are validated for several laboratory applications, with the most common being Western Blot (WB) and Indirect ELISA . Some vendors offer antibody pairs specifically designed for immunoprecipitation followed by Western blot detection (IP-WB) . These paired antibodies typically include one antibody (often rabbit polyclonal) for immunoprecipitation and another (mouse polyclonal) for detection in Western blot . For comprehensive protein interaction studies, researchers should select antibodies validated for multiple applications to ensure consistent results across experimental platforms.

What is the molecular profile of MAD2L1BP protein?

MAD2L1BP has both calculated and observed molecular weights of 34 kDa . The protein is encoded by the gene with NCBI Gene ID 9587 and corresponds to UNIPROT ID Q15013 . The gene symbol is MAD2L1BP, with alternative aliases including CMT2, KIAA0110, and MGC11282 . This information is critical for researchers when validating antibody specificity through Western blot and ensuring they have correctly identified the target protein.

What criteria should researchers use when selecting a MAD2L1BP antibody?

When selecting a MAD2L1BP antibody, researchers should consider:

Researchers studying MAD2L1BP mutations should particularly note that antibodies targeting the C-terminal region may not detect truncated variants, such as those resulting from the p.R285* mutation .

How can researchers validate MAD2L1BP antibody specificity?

Validation of MAD2L1BP antibody specificity requires multiple complementary approaches:

• Positive Controls: Use cell lines or tissues known to express MAD2L1BP. Human cell lines are appropriate as they express detectable levels of the protein .

• Negative Controls: Implementation of MAD2L1BP knockdown (siRNA) or knockout models. While challenging in oocytes (as siRNAs targeting MAD2L1BP have shown limited effectiveness in oocyte models), F9 cell lines have demonstrated successful knockdown with approximately 70% reduction in MAD2L1BP levels .

• Molecular Weight Verification: Confirm the detection of a 34 kDa band in Western blot applications, which corresponds to the expected size of MAD2L1BP .

• Cross-Antibody Validation: Compare results using antibodies targeting different epitopes of MAD2L1BP to ensure consistent detection.

• Peptide Competition Assays: Pre-incubate the antibody with purified MAD2L1BP protein or immunizing peptide to demonstrate binding specificity.

How can MAD2L1BP antibodies be used to study spindle assembly checkpoint dynamics?

MAD2L1BP antibodies enable detailed investigation of spindle assembly checkpoint (SAC) dynamics through multiple experimental approaches:

• Co-Immunoprecipitation Studies: Using IP-WB antibody pairs to isolate MAD2L1BP-containing complexes and identify interaction partners such as MAD2L1 and TRIP13 . This approach has revealed that MAD2L1BP functions as an adaptor that tightly binds MAD2 and recruits TRIP13 .

• Cell Cycle Synchronization Analysis: Western blotting of synchronized cell populations to monitor changes in MAD2L1BP expression and post-translational modifications throughout the cell cycle.

• Live Cell Imaging: Combined with fluorescently tagged proteins to visualize MAD2L1BP dynamics during mitotic progression.

• Rescue Experiments: As demonstrated in oocyte studies, where wild-type MAD2L1BP cRNA co-injection with MAD2 cRNA allowed progression beyond meiosis I, while mutant MAD2L1BP failed to rescue the MAD2-induced arrest .

These approaches collectively provide insights into how MAD2L1BP coordinates the inactivation of the spindle checkpoint to facilitate cell cycle progression.

What experimental models are suitable for studying MAD2L1BP function?

Several experimental models have proven effective for MAD2L1BP functional studies:

• Oocyte Models: Both human and mouse oocytes express high levels of MAD2L1BP mRNA, particularly at advanced maturation stages . Mouse oocyte models have been successfully used for microinjection of MAD2 and MAD2L1BP cRNAs to study meiotic progression .

• Cell Line Models: Human cell lines are suitable for protein expression studies, while mouse F9 cells have been used successfully for siRNA-mediated knockdown of MAD2L1BP .

• Minigene Assay Systems: Effective for studying splicing variants, such as those resulting from the c.21-94G>A variant, using HEK293T cells transfected with minigene constructs .

• MAD2 Overexpression Models: Overexpression of MAD2 in GV (germinal vesicle) oocytes induces meiotic MI arrest, providing a controlled system to assess MAD2L1BP function through rescue experiments .

How can researchers investigate MAD2L1BP mutations and their impact on fertility?

Recent discoveries have linked MAD2L1BP mutations to female infertility, offering important research directions:

• Genetic Screening: Identify biallelic variants in MAD2L1BP in patients with primary infertility, focusing on homozygous and compound heterozygous variants .

• mRNA Expression Analysis: Quantitative PCR to assess how variants affect MAD2L1BP mRNA expression levels .

• Minigene Splicing Assays: To evaluate how intronic variants (such as c.21-94G>A) affect splicing patterns and potentially create premature termination codons .

• Functional Protein Studies: Assess protein-protein interactions, particularly MAD2L1BP binding to MAD2, using wild-type and mutant proteins .

• Oocyte Maturation Assays: Monitor polar body extrusion in oocytes expressing wild-type versus mutant MAD2L1BP to assess meiotic progression .

• Transcriptome Analysis: Compare gene expression profiles between normal and MAD2L1BP-mutant oocytes to identify downstream effects, particularly on mitotic checkpoint complex components .

What are the optimal conditions for Western blot analysis using MAD2L1BP antibodies?

For reliable Western blot detection of MAD2L1BP, researchers should follow these optimized protocols:

When examining mutant forms of MAD2L1BP, researchers should be aware that C-terminal truncations (like p.R285*) may still be detected at approximately 34 kDa but lack functional capacity to bind MAD2 .

What strategies can overcome challenges in MAD2L1BP immunoprecipitation?

Successful immunoprecipitation of MAD2L1BP and its interaction partners requires specific technical considerations:

• Antibody Selection: Use antibody pairs specifically designed for IP-WB applications, with separate antibodies for immunoprecipitation and detection .

• Lysis Conditions: Utilize gentle lysis buffers that preserve protein-protein interactions. For MAD2L1BP-MAD2 interactions, NP-40-based buffers with protease inhibitors are recommended.

• Cross-linking Option: For transient or weak interactions, consider reversible cross-linking approaches to stabilize complexes before immunoprecipitation.

• Bead Selection: Protein A Magnetic Beads have been successfully used for MAD2L1BP immunoprecipitation .

• Controls: Include IgG control immunoprecipitations and input samples to verify specificity of detected interactions.

• Detection Strategy: After immunoprecipitation with rabbit polyclonal anti-MAD2L1BP, detection with mouse purified polyclonal anti-MAD2L1BP has proven effective .

How should researchers approach MAD2L1BP functional rescue experiments?

Functional rescue experiments, particularly in oocyte models, require careful experimental design:

• cRNA Preparation: In vitro transcription from linearized plasmids containing wild-type or mutant MAD2L1BP sequences .

• Microinjection Protocol: Co-inject MAD2 cRNAs (to induce MI arrest) with either wild-type or mutant MAD2L1BP cRNAs into GV stage oocytes .

• Culture Conditions: Extended culture in M16 medium to allow sufficient time for meiotic progression .

• Phenotypic Assessment: Monitor polar body extrusion (PBE) as the primary readout for progression beyond meiosis I .

• Controls: Include oocytes injected with MAD2 cRNA alone as positive controls for MI arrest .

• Quantification: Compare the percentage of oocytes with PBE between wild-type and mutant MAD2L1BP groups .

This approach has successfully demonstrated that while wild-type MAD2L1BP can rescue MAD2-induced MI arrest, the p.R285* mutant fails to do so, confirming its functional deficiency .

What are emerging areas for MAD2L1BP antibody applications in research?

Several promising research directions could benefit from MAD2L1BP antibodies:

• Clinical Biomarker Development: Investigating MAD2L1BP as a potential biomarker for female infertility conditions related to oocyte maturation defects.

• Therapeutic Target Validation: Using antibodies to validate MAD2L1BP as a potential therapeutic target for conditions involving dysregulated cell cycle control.

• Single-Cell Analysis: Applying MAD2L1BP antibodies in single-cell proteomics to understand cell-to-cell variability in spindle checkpoint regulation.

• Structure-Function Relationships: Combining antibody-based approaches with structural biology techniques to further elucidate how MAD2L1BP's domains contribute to its function.

• Post-Translational Modification Mapping: Developing modification-specific antibodies to understand how MAD2L1BP activity is regulated throughout the cell cycle.

These emerging applications highlight the continuing importance of MAD2L1BP antibodies as versatile tools in both basic and translational research.