MCM9 Monoclonal Antibody

What is MCM9 and what are its primary cellular functions?

MCM9 is a member of the minichromosome maintenance (MCM) protein family, known to be essential for the initiation of DNA replication. Unlike other MCM family members, MCM9 has specialized functions in DNA repair rather than primary DNA replication. MCM9 functions primarily as a component of the MCM8-MCM9 complex, which is involved in the repair of double-stranded DNA breaks (DSBs) and DNA interstrand cross-links (ICLs) through homologous recombination (HR) .

The MCM8-MCM9 complex contributes to DNA repair through multiple mechanisms:

• Recruiting the MRE11-RAD50-NBN/NBS1 (MRN) complex to DNA damage sites

• Promoting nuclease activity in DNA resection

• Indirectly regulating the recruitment of the recombinase RAD51 to damage sites

• Acting as a helicase in DNA mismatch repair (MMR) following DNA replication errors

• Recruiting MLH1, a component of the MMR complex, to chromatin

Interestingly, the MCM8-MCM9 complex is dispensable for DNA replication and S phase progression, distinguishing it from other MCM family members .

What is the structure of MCM9 and how does it relate to its function?

MCM9 proteins in humans consist of 1143 amino acids with a molecular weight of approximately 127-130 kDa . The protein contains several functional domains, including a DNA-binding domain and an ATPase domain characteristic of helicases.

MCM9 forms a complex with MCM8, which recent structural studies have shown to potentially form a hexameric structure that explains its function as a DNA helicase during homologous recombination . The protein contains critical N-C linker regions that are essential for its cellular resistance to DNA-damaging reagents. Deletion of these linker regions significantly reduces the protein's ability to confer resistance to cisplatin, a DNA cross-linking agent that induces damage primarily repaired by homologous recombination .

The central region of MCM9 contains the conserved MCM box, which is the catalytic core with ATPase activity essential for its helicase function. This structure enables MCM9 to participate in unwinding DNA during repair processes.

What are the key considerations when selecting an MCM9 monoclonal antibody for research?

When selecting an MCM9 monoclonal antibody, researchers should consider:

• Specificity: Ensure the antibody specifically recognizes MCM9 without cross-reactivity to other MCM family proteins. Review antibody validation data showing specificity testing .

• Applications: Verify the antibody has been validated for your specific application (Western blot, immunoprecipitation, immunofluorescence, etc.). For example, some antibodies may be validated for WB but not for IP or IHC .

• Species reactivity: Confirm the antibody recognizes MCM9 in your species of interest. Available antibodies show reactivity with human, mouse, and rat samples .

• Epitope location: Consider the epitope recognized by the antibody. Some antibodies target the N-terminal region (aa 1-400), which may affect detection if your research involves N-terminal modifications or truncations .

• Validation data: Review published literature using the antibody and examine manufacturer validation data, including positive and negative controls.

• Isotype and host species: Consider whether a polyclonal or monoclonal antibody better suits your needs, and whether the host species (rabbit, mouse, etc.) is compatible with your experimental design .

How should MCM9 antibodies be validated for research applications?

Comprehensive validation of MCM9 antibodies should follow these methodological steps:

• Structural integrity testing: Use techniques like SDS-PAGE, isoelectric focusing (IEF), HPLC, or mass spectrometry to confirm the antibody is not fragmented, aggregated, or otherwise modified .

• Specificity validation:

  • Direct binding assays with positive and negative controls

  • Use isotype-matched, irrelevant antibodies as negative controls

  • Test against negative antigen controls (similar chemical nature but antigenically unrelated)

• Cross-reactivity assessment: Test against other MCM family proteins, particularly MCM8 given their close association.

• Functional validation:

  • Confirm detection of native and denatured MCM9 if applicable to the application

  • Validate antibody function in all claimed applications (WB, IP, IF, ELISA, etc.)

  • Perform knockdown/knockout controls when possible

• Epitope characterization: When possible, define the specific epitope recognized by the antibody, especially if studying specific domains of MCM9 .

• Potency assays: Quantify antibody binding activity through affinity, avidity, or immunoreactivity measurements appropriate to the intended application .

How can MCM9 antibodies be used to study DNA damage repair pathways?

MCM9 antibodies can be employed in multiple experimental approaches to study DNA damage repair:

• Colocalization studies: Use immunofluorescence with MCM9 antibodies to track localization of MCM9 to DNA damage sites after inducing damage with agents like cisplatin. This can be combined with antibodies against other repair factors like RAD51 or the MRN complex to study recruitment dynamics .

• Chromatin association studies: Use chromatin fractionation followed by Western blotting with MCM9 antibodies to quantify MCM9 recruitment to chromatin after DNA damage .

• Co-immunoprecipitation: Use MCM9 antibodies for co-IP experiments to identify interaction partners in the DNA damage response pathway, such as MCM8, components of the MRN complex, or HROB/MCM8IP .

• ChIP-seq approaches: Combine MCM9 antibodies with ChIP-seq to map genome-wide binding sites after DNA damage, revealing preferential sites of action.

• Recruitment kinetics: Use live-cell imaging with fluorescently tagged MCM9 antibodies (or fragments) to study the temporal dynamics of MCM9 recruitment to damage sites.

• Functional rescue experiments: In MCM9 knockout/knockdown systems, use wildtype versus mutant MCM9 complementation followed by antibody detection to assess functional domains .

What experimental approaches can determine if MCM9 functions as predicted in cellular models?

To experimentally validate MCM9 function in cellular models:

• Chemoresistance assays: Treat cells with DNA-damaging agents like cisplatin and measure survival in MCM9 wildtype versus knockout/knockdown cells. Complete or partial complementation with wildtype MCM9 should restore resistance, while mutant versions (e.g., N-C linker deletions) may fail to complement .

• DNA damage foci analysis: Induce DNA damage and quantify repair foci (γH2AX, RAD51) in cells with normal versus disrupted MCM9 function.

• HR reporter assays: Use established homologous recombination reporter systems (such as DR-GFP) to measure HR efficiency in the presence or absence of functional MCM9.

• Helicase activity assays: Use purified MCM8-MCM9 complex in biochemical assays to measure ATP-dependent DNA unwinding activity on various DNA substrates.

• Synthetic lethality screening: Combine MCM9 deficiency with deficiencies in other DNA repair pathways to identify genetic interactions and backup repair mechanisms.

• CRISPR-Cas9 mutagenesis: Generate specific domain mutations in MCM9 to test structure-function relationships and detect the protein using domain-specific antibodies.

Why might MCM9 antibodies show inconsistent results in immunofluorescence studies?

Inconsistent results in immunofluorescence studies with MCM9 antibodies may result from several factors:

• Cell cycle dependence: MCM9 localization and expression levels may vary throughout the cell cycle. Synchronize cells or co-stain with cell cycle markers to control for this variable.

• DNA damage induction variability: MCM9 recruitment to damage sites depends on the type and extent of DNA damage. Standardize damage induction protocols with appropriate positive controls.

• Fixation and permeabilization sensitivity: MCM9 epitopes may be sensitive to specific fixation methods. Test multiple fixation protocols (paraformaldehyde, methanol, etc.) and permeabilization conditions.

• Antibody batch variation: Different lots of antibody may have varying specificities. Always include known positive controls with each new lot.

• Blocking conditions: Optimize blocking buffers to reduce background while maintaining specific signal. BSA, serum, or commercial blocking reagents may perform differently.

• Epitope masking: MCM9 interactions with other proteins might mask antibody epitopes. Consider antigen retrieval methods if appropriate for your samples.

• Antibody concentration: Titrate antibody to determine optimal concentration for your specific application and cell type.

How can specificity issues with MCM9 antibodies be addressed in Western blotting?

To address specificity issues with MCM9 antibodies in Western blotting:

• Include proper controls:

  • Positive control (cell line known to express MCM9)

  • Negative control (MCM9 knockout/knockdown cells)

  • Loading control to normalize protein amounts

• Optimize blocking conditions: Test different blocking agents (non-fat dry milk, BSA, commercial blockers) to reduce non-specific binding.

• Adjust antibody concentration: Titrate primary antibody concentration to find the optimal signal-to-noise ratio.

• Increase washing stringency: More stringent or frequent washing steps can help reduce non-specific binding.

• Use alternative antibodies: If available, test antibodies that recognize different epitopes of MCM9 to confirm specificity.

• Pre-adsorption: Pre-adsorb the antibody with the immunizing peptide (if available) to confirm specificity.

• Optimize protein loading: Adjust total protein amounts to ensure detection within the linear range of the antibody.

• Denaturing conditions: Modify sample preparation (reducing vs. non-reducing, boiling time) as MCM9's large size (127 kDa) may affect transfer and detection efficiency .

How are MCM9 antibodies being used to study reproductive disorders?

MCM9 antibodies are instrumental in studying reproductive disorders, particularly in ovarian insufficiency models:

• Ovarian tissue analysis: In studies of primary ovarian insufficiency (POI), MCM9 antibodies can be used for immunohistochemistry to examine MCM9 expression patterns in ovarian tissues from affected versus unaffected individuals .

• Genotype-phenotype correlations: After identifying MCM9 variants in patients with reproductive disorders, antibodies can detect alterations in protein expression, stability, or localization resulting from these variants .

• Animal model validation: In MCM9 knockout mice, which exhibit sterility and gametogenesis deficiencies, antibodies help characterize the cellular and molecular consequences of MCM9 deficiency in reproductive tissues .

• Mechanistic studies: Using cellular models of identified patient mutations, antibodies can track how mutant MCM9 proteins affect DNA repair pathways in reproductive cell types.

• Biomarker development: Research is evaluating whether MCM9 expression levels or localization patterns, detected via antibodies, could serve as biomarkers for susceptibility to certain reproductive disorders.

The connection between MCM9 and reproductive health stems from findings that MCM9 knockout mice show gametogenesis defects and that MCM9 variants have been identified in women with primary ovarian insufficiency, highlighting its importance in reproductive cell development and function .

What role does MCM9 play in cancer research, and how are antibodies utilized in this field?

MCM9's function in DNA repair pathways makes it relevant to cancer research in several aspects:

• Chemoresistance mechanisms: MCM9 antibodies help study how cancer cells with altered MCM9 expression respond to DNA-damaging chemotherapeutics like cisplatin. Resistance to cisplatin has been linked to MCM9 function in repairing DNA interstrand crosslinks .

• Homologous recombination deficiency: In tumors with HR deficiencies (like BRCA-mutated cancers), MCM9 may function in compensatory repair pathways. Antibodies help characterize these alternative pathways.

• Expression profiling: Immunohistochemistry with MCM9 antibodies can assess expression levels across tumor types and correlate with progression or treatment response.

• Synthetic lethality screening: MCM9 antibodies assist in validating knockdown efficiency in screens seeking genetic interactions that could be therapeutically exploited.

• PARP inhibitor response studies: Since HR is crucial for resistance to PARP inhibitors, MCM9 antibodies help investigate whether MCM9 status affects response to these targeted therapies.

• Genomic instability assessment: Antibodies against MCM9 and its interaction partners provide tools to study genomic instability mechanisms in cancer cells with DNA repair defects.

How can MCM9 antibodies be used to investigate the structural dynamics of the MCM8-MCM9 complex?

Recent structural insights into the MCM8-MCM9 complex offer opportunities for more sophisticated antibody applications:

• Conformational antibodies: Developing antibodies that recognize specific conformational states of the MCM8-MCM9 complex could reveal structural changes during activation.

• Domain-specific antibodies: Antibodies targeting specific domains (like the N-C linker regions) can investigate structure-function relationships, particularly since these regions are critical for cisplatin resistance .

• Super-resolution microscopy: Combining domain-specific antibodies with super-resolution techniques can map the spatial organization of MCM8-MCM9 complexes at DNA damage sites.

• Single-molecule studies: Using fluorescently labeled antibody fragments in single-molecule studies can track conformational changes in real-time.

• Cryo-EM sample validation: Antibodies can verify the integrity and composition of MCM8-MCM9 samples prepared for structural studies.

• Hexameric assembly investigation: Since recent research suggests a hexameric structure for MCM8-MCM9 similar to other MCM complexes, antibodies could be used to probe this assembly in cells under different conditions .

• HROB interaction studies: Antibodies against MCM8-MCM9 and HROB can investigate how HROB facilitates loading of the MCM8-MCM9 complex onto DNA and stimulates its helicase activity .

What novel approaches are being developed to study MCM9's role in maintaining genomic stability?

Emerging approaches to study MCM9's role in genomic stability include:

• CRISPR-based DNA damage recruitment assays: Using CRISPR to create site-specific DNA damage and tracking MCM9 recruitment via antibodies in real-time.

• Proximity labeling techniques: BioID or APEX2 fusion proteins combined with MCM9 antibodies to identify the damage-specific interactome of MCM9.

• Patient-derived organoids: Studying MCM9 function in 3D organoid cultures from patients with MCM9 variants, using antibodies to track protein behavior in a more physiological context.

• Single-cell approaches: Combining single-cell sequencing with antibody-based detection to understand cell-to-cell variability in MCM9 function during DNA damage response.

• Optical tweezers and DNA curtains: Using purified components and antibodies to study mechanistic aspects of MCM8-MCM9 helicase activity on single DNA molecules.

• Synthetic genetic array analysis: Systematic genetic interaction mapping combined with antibody validation to place MCM9 in the broader DNA repair network.

• Liquid-liquid phase separation (LLPS) studies: Investigating whether MCM9 participates in phase-separated repair foci using antibodies to track these dynamics.

What are the optimal conditions for immunoprecipitation of MCM9?

For successful immunoprecipitation of MCM9:

• Lysis buffer optimization:

  • Use buffers containing 150-300 mM NaCl, 1% NP-40 or Triton X-100, 50 mM Tris-HCl (pH 7.4-8.0)

  • Include protease inhibitors, phosphatase inhibitors, and potentially 1-2 mM ATP to stabilize the complex

  • For nuclear proteins like MCM9, consider specialized nuclear extraction protocols

• Antibody selection and amount:

  • Use antibodies validated specifically for IP applications

  • Typically use 2-5 μg antibody per 500 μg-1 mg of total protein lysate

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Incubation conditions:

  • Overnight incubation at 4°C with gentle rotation often yields best results

  • Avoid harsh detergents that may disrupt protein-protein interactions

• Washing stringency:

  • Balance between removing non-specific interactions and maintaining specific ones

  • Consider graduated washing with increasing salt concentrations

• Elution methods:

  • For downstream applications like mass spectrometry, native elution with peptide competition

  • For Western blot, direct elution in SDS sample buffer

• Co-factor considerations:

  • Include ATP or non-hydrolyzable analogs if studying the intact MCM8-MCM9 complex

  • Consider including DNA if studying DNA-bound complexes

• Control IPs:

  • Always perform parallel IPs with isotype-matched irrelevant antibodies

  • Include input, unbound, and IP fractions in analyses

How should samples be prepared for optimal detection of MCM9 in Western blotting?

For optimal detection of MCM9 in Western blotting:

• Sample preparation:

  • For full-length MCM9 (127 kDa), use fresh samples whenever possible

  • Incorporate protease inhibitors to prevent degradation

  • If studying chromatin-bound MCM9, include a fractionation protocol to separate chromatin-bound from soluble protein

• Denaturation conditions:

  • Heat samples at 95°C for 5 minutes in standard Laemmli buffer with β-mercaptoethanol

  • For detection of certain complexes or conformations, consider milder denaturation (70°C for 10 minutes)

• Gel selection:

  • Use lower percentage gels (6-8%) or gradient gels to resolve the 127 kDa MCM9 protein effectively

  • Consider using pre-cast commercial gels for consistent results

• Transfer optimization:

  • For large proteins like MCM9, use wet transfer (preferably overnight at low voltage)

  • Add 0.1% SDS to transfer buffer to facilitate transfer of large proteins

  • Consider using PVDF membrane (0.45 μm pore size) rather than nitrocellulose

• Blocking conditions:

  • Test both BSA and non-fat dry milk to determine optimal blocking

  • Typically 5% blocking agent in TBST for 1 hour at room temperature

• Antibody dilution:

  • Primary antibody dilutions typically range from 1:500 to 1:2000

  • Optimize through titration experiments

  • Incubate overnight at 4°C for best results

• Loading controls:

  • Use appropriate high-molecular-weight loading controls

  • Consider multiple loading controls for verification

• Signal detection:

  • Enhanced chemiluminescence (ECL) systems work well

  • For quantitative analysis, consider fluorescent secondary antibodies and imaging