CMT2 Antibody

Basic Research Questions

• What is the target of Anti-CMT2 Antibody and what is its biological significance?

The Anti-CMT2 Antibody [ARC2187] specifically targets MAD2L1 Binding Protein (MAD2L1BP), a 37 kDa protein involved in mitotic checkpoint regulation . Methodologically, understanding this target is essential for designing appropriate experimental controls. MAD2L1BP functions as a negative regulator of the spindle assembly checkpoint by interacting with MAD2, promoting mitotic progression. When designing experiments to study cell cycle regulation, researchers should consider the temporal expression patterns of this protein throughout mitosis and its interactions with other checkpoint proteins.

• What applications is the Anti-CMT2 Antibody validated for?

The Anti-CMT2 Antibody [ARC2187] has been validated for multiple research applications with specific methodological parameters :

• Western Blot (WB): Recommended dilutions of 1:500-1:2,000

• Immunohistochemistry (IHC): Recommended dilutions of 1:50-1:200

• Immunocytochemistry/Immunofluorescence (ICC/IF): Recommended dilutions of 1:50-1:200

For each application, researchers should begin with the recommended dilutions and optimize based on their specific experimental system. Initial validation should include positive controls expressing known levels of MAD2L1BP and appropriate negative controls.

• What species reactivity should be considered when using the Anti-CMT2 Antibody?

The Anti-CMT2 Antibody [ARC2187] demonstrates reactivity with human, mouse, and rat samples . This cross-species reactivity is methodologically advantageous for comparative studies. Researchers should verify antibody performance in their specific model system through preliminary experiments. When designing cross-species studies, consider the sequence homology of the target epitope region (amino acids 175-274 of human MAD2L1BP ) across different species to anticipate potential variations in binding affinity and specificity.

• What are the recommended storage and handling protocols for maximizing Anti-CMT2 Antibody performance?

To maintain optimal antibody performance, follow these methodological considerations :

• Shipping temperature: The antibody is shipped at 4°C

• Long-term storage: Store at -20°C upon receipt

• Aliquoting: Divide into single-use aliquots to avoid repeated freeze/thaw cycles

• Formulation stability: The antibody is supplied in Phosphate Buffered Saline (pH 7.3) with 50% Glycerol, 0.05% BSA, and 0.02% Sodium Azide

This formulation maintains antibody stability during storage, but researchers should avoid more than 3-5 freeze/thaw cycles, as this can significantly decrease antibody performance through protein denaturation and aggregation.

• What controls should be included when using Anti-CMT2 Antibody in research protocols?

Methodologically rigorous experiments with Anti-CMT2 Antibody should include these controls :

• Isotype controls: Rabbit IgG (A82272 or A17360) at matching concentrations

• Positive controls: Cell lines or tissues with confirmed MAD2L1BP expression

• Negative controls: Primary antibody omission and ideally MAD2L1BP-knockdown samples

• Loading controls: For Western blots, include housekeeping proteins (β-actin recommended)

These controls allow researchers to distinguish specific from non-specific binding and validate the sensitivity and specificity of the detection system.

Advanced Research Questions

• How can researchers validate the specificity of Anti-CMT2 Antibody in their experimental systems?

A comprehensive validation strategy should include:

• Gene silencing approach: Compare antibody signal between wild-type and MAD2L1BP-depleted cells (siRNA or CRISPR)

• Recombinant protein detection: Test antibody against purified MAD2L1BP protein

• Peptide competition: Pre-incubate antibody with the immunizing peptide (amino acids 175-274 )

• Cross-reactivity assessment: Test against closely related proteins in the MAD2L1 interactome

• Multiple detection methods: Confirm findings using alternative techniques (e.g., mass spectrometry)

This multi-parameter validation approach establishes confidence in antibody specificity and ensures experimental reproducibility.

• What are the optimal fixation and permeabilization conditions for detecting MAD2L1BP in different sample types?

The methodological approach to fixation varies by application:

For IHC:

• Formalin-fixed paraffin-embedded (FFPE) tissues: Perform heat-mediated antigen retrieval

• Frozen sections: 4% paraformaldehyde fixation followed by gentle permeabilization

For ICC/IF:

• Adherent cells: 4% paraformaldehyde (10-15 minutes) followed by 0.1-0.25% Triton X-100 permeabilization

• Suspension cells: Methanol fixation/permeabilization (-20°C, 10 minutes)

The rabbit monoclonal nature of Anti-CMT2 Antibody [ARC2187] makes it compatible with most standard fixation protocols, but optimization is essential as overfixation can mask the epitope (amino acids 175-274).

• What strategies can researchers employ when troubleshooting weak or absent signals with Anti-CMT2 Antibody?

A methodological troubleshooting approach includes:

Each troubleshooting step should be systematically documented to establish optimal conditions for specific experimental systems.

• How can Anti-CMT2 Antibody be used in co-immunoprecipitation studies to investigate MAD2L1BP protein interactions?

For co-immunoprecipitation (Co-IP) of MAD2L1BP complexes:

• Lysis methodology: Use gentle, non-denaturing lysis buffers (e.g., 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40) with protease/phosphatase inhibitors

• Pre-clearing: Incubate lysate with Protein A/G beads to reduce non-specific binding

• Immunoprecipitation: Incubate pre-cleared lysate with Anti-CMT2 Antibody (3-5 μg per mg of protein)

• Complex capture: Add Protein A beads (compatible with rabbit IgG )

• Washing: Perform stringent washes to remove non-specific interactions

• Elution and analysis: SDS-PAGE separation followed by immunoblotting for interacting partners

This methodological approach can identify novel MAD2L1BP protein interactions and validate known associations with MAD2 and other mitotic checkpoint components.

• How does phosphorylation state impact MAD2L1BP detection with Anti-CMT2 Antibody?

The Anti-CMT2 Antibody [ARC2187] targets amino acids 175-274 of human MAD2L1BP , a region that contains potential phosphorylation sites that may affect epitope accessibility. Methodologically, researchers should consider:

• Phosphatase treatment: Compare antibody signal with and without lambda phosphatase treatment

• Cell cycle synchronization: Analyze MAD2L1BP detection across different cell cycle phases

• Phospho-specific detection: Combine with phospho-specific antibodies in multiplexed experiments

• Mobility shift analysis: Examine band patterns for shifts indicating post-translational modifications

Understanding the impact of phosphorylation on antibody binding is crucial for correctly interpreting experimental results, especially in studies of mitotic regulation where phosphorylation dynamics are prominent.

• What methodologies are recommended for quantitative analysis of MAD2L1BP expression using Anti-CMT2 Antibody?

For rigorous quantitative analysis:

• Western blot quantification:

  • Use gradient gels for optimal separation

  • Include titrated recombinant MAD2L1BP standards

  • Verify linear detection range with the recommended 1:500-1:2,000 dilution

  • Employ fluorescent secondary antibodies for wider linear detection range

• Immunofluorescence quantification:

  • Use consistent exposure settings across all samples

  • Include intensity calibration standards

  • Apply automated image analysis with defined thresholds

  • Perform Z-stack acquisition to capture total cellular expression

• Flow cytometry analysis:

  • Optimize fixation/permeabilization for intracellular staining

  • Use fluorophore-conjugated secondary antibodies with minimal spectral overlap

  • Include appropriate compensation controls

  • Analyze median fluorescence intensity rather than mean values

These methodological approaches enable reliable quantification of MAD2L1BP expression across experimental conditions.

• How can researchers design multiplexed experiments using Anti-CMT2 Antibody with other cell cycle markers?

For effective multiplexed detection:

• Antibody selection considerations:

  • Choose antibodies raised in different host species to avoid cross-reactivity

  • Verify that the rabbit monoclonal Anti-CMT2 Antibody [ARC2187] is compatible with other primary antibodies

  • Select antibodies targeting proteins with distinct subcellular localizations

• Sequential staining methodology:

  • Begin with the lowest concentration antibody

  • Consider tyramide signal amplification for sequential detection

  • Use spectral unmixing for closely overlapping fluorophores

• Analytical approach:

  • Employ colocalization analysis to examine spatial relationships

  • Quantify correlation coefficients between MAD2L1BP and other markers

  • Analyze co-expression patterns throughout the cell cycle

This methodological framework enables complex experimental designs to study MAD2L1BP in relation to other cell cycle regulatory proteins in a spatiotemporal context.