MCM9 Antibody, HRP conjugated

What is MCM9 and what cellular functions does it serve in DNA repair mechanisms?

MCM9 is a component of the MCM8-MCM9 complex that plays critical roles in homologous recombination (HR) repair following DNA interstrand cross-links (ICLs) and double-stranded DNA breaks (DSBs) . The protein functions as a DNA helicase that acts downstream of Fanconi anemia proteins BRCA2 and RAD51 . Its primary roles include:

• Processing aberrant replication forks into homologous recombination substrates

• Orchestrating homologous recombination through coordinated activities involving resection, fork stabilization, and fork restart

• Contributing to DNA mismatch repair by unwinding mismatched DNA strands

• Recruiting repair proteins like MLH1 to chromatin

• Facilitating gametogenesis through its role in maintaining genome integrity

The MCM8-MCM9 complex is dispensable for DNA replication and S phase progression but critical for repair processes . Loss of MCM9 leads to impaired HR-mediated fork rescue due to decreased recruitment of the MRN helicase/nuclease complex, RAD51 recombinase, and RPA single-stranded DNA binding protein after DNA damage .

How should MCM9 Antibody, HRP conjugated be stored and handled to maintain optimal activity?

For proper storage and handling of MCM9 Antibody, HRP conjugated:

• Transport and initial receipt: The antibody is typically shipped at 4°C .

• Short-term storage: Upon delivery, aliquot and store at -20°C .

• Long-term storage: For extended preservation, store at -80°C .

• Avoid repeated freeze-thaw cycles as this can degrade antibody activity and compromise the HRP conjugate .

• Buffer conditions: The antibody is typically supplied in 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4 buffer to maintain stability .

This methodological approach to storage is essential for maintaining both the binding specificity of the antibody and the enzymatic activity of the HRP conjugate, which is critical for detection in experimental applications.

What experimental applications are most suitable for MCM9 Antibody, HRP conjugated?

For optimal results in detecting native MCM9 protein, researchers should consider that:

• The antibody recognizes human and mouse MCM9 protein with high specificity

• The immunogen used for antibody production typically corresponds to amino acids 1-391 of the human DNA helicase MCM9 protein

• Purity levels are generally ≥95% following protein G chromatography purification

What controls are essential when working with MCM9 Antibody, HRP conjugated?

When designing experiments using MCM9 Antibody, HRP conjugated, implement these methodological controls:

• Positive controls:

  • Cell lines known to express MCM9 (most proliferating cells express basal levels)

  • Cells treated with DNA damaging agents that induce MCM9 recruitment (e.g., mitomycin C or cisplatin)

• Negative controls:

  • MCM9 knockout cells generated through CRISPR/Cas9 methods

  • Isotype control (rabbit IgG-HRP with matching concentration)

  • Secondary antibody-only controls (for non-direct detection methods)

• Specificity controls:

  • Pre-absorption with the immunizing peptide (amino acids 1-391)

  • Comparative detection using alternative MCM9 antibodies raised against different epitopes

• Technical controls:

  • HRP activity control to ensure enzymatic function of the conjugate

  • Serial dilution series to confirm signal linearity and quantitation range

These controls are particularly important when investigating MCM9's dynamic localization to sites of DNA damage, as false positives can easily occur in complex nuclear repair foci.

How can MCM9 Antibody, HRP conjugated be used to investigate homologous recombination pathways?

To investigate homologous recombination (HR) pathways using MCM9 Antibody, HRP conjugated, researchers can employ several sophisticated methodological approaches:

• Chromatin fractionation assays:

  • Separate chromatin-bound and soluble nuclear fractions following DNA damage

  • Quantify MCM9 recruitment to chromatin using the HRP-conjugated antibody in ELISA format

  • Compare recruitment kinetics in wild-type versus HR-deficient backgrounds (e.g., BRCA2, RAD51 knockdowns)

• Proximity ligation assays (PLA):

  • Combine MCM9 Antibody with antibodies against known HR factors (e.g., RAD51, BRCA2)

  • Use the HRP-conjugated MCM9 antibody as one component of the PLA system

  • Quantify protein-protein interactions at sites of DNA damage

• Sequential ChIP approaches:

  • First IP with antibodies against γH2AX or other DNA damage markers

  • Follow with MCM9 Antibody, HRP conjugated detection

  • Analyze co-localization at specific genomic loci

Research findings indicate that MCM9 acts alongside HROB in promoting homologous recombination downstream of RAD51 . In cells lacking both HROB and the HELQ helicase, HR is severely impaired, suggesting MCM9 functions in one of two major pathways for HR completion .

What methodological considerations are important when studying MCM9's role in DNA fork stability and progression?

When investigating MCM9's functions in replication fork stability, implement these methodological approaches:

• DNA fiber analysis:

  • Pulse-label cells with nucleoside analogs (e.g., CldU followed by IdU)

  • Induce replication stress during the second label (e.g., HU, cisplatin)

  • Compare fork progression rates and restart efficiency in MCM9-proficient versus deficient cells

  • Detect MCM9 at stalled forks using the HRP-conjugated antibody combined with immunofluorescence detection of labeled DNA

• Electron microscopy analysis of replication intermediates:

  • Extract and spread genomic DNA from cells with and without MCM9

  • Visualize replication fork structures (e.g., reversed forks, chicken foot structures)

  • Quantify abnormal replication intermediates

• iPOND (isolation of Proteins On Nascent DNA) coupled with ELISA:

  • Label nascent DNA with EdU

  • Crosslink proteins to DNA and perform click chemistry

  • Use MCM9 Antibody, HRP conjugated in ELISA format to quantify MCM9 association with nascent DNA before and after fork stalling

Evidence shows that MCM9 is required for DNA resection by recruiting the MRE11-RAD50-NBN/NBS1 (MRN) complex to repair sites and promoting its nuclease activity . This function is critical for processing stalled forks into HR substrates and likely contributes to fork stability during replication stress.

How can researchers distinguish between MCM9's role in homologous recombination versus mismatch repair?

To differentiate between MCM9's distinct functions in homologous recombination (HR) and mismatch repair (MMR), employ these methodological approaches:

• Functional complementation assays:

  • Generate cells expressing MCM9 mutants with lesion-specific defects

  • Test via selective DNA damage induction:

    • Mitomycin C (MMC) or cisplatin for interstrand crosslinks (HR pathway)

    • Base mismatch inducers like 6-TG for MMR pathway

  • Quantify recruitment of MCM9 to different types of lesions using the HRP-conjugated antibody

• Co-immunoprecipitation coupled with ELISA detection:

  • Immunoprecipitate with antibodies against HR-specific proteins (RAD51, BRCA2) or MMR-specific proteins (MLH1, MSH2)

  • Detect co-precipitated MCM9 using the HRP-conjugated antibody in ELISA format

  • Compare interaction profiles under different damage conditions

• Domain-specific functional analysis:

  • The MCM9 C-terminal extension (CTE) contains specific motifs that direct localization to DNA damage sites

  • A variant BRC motif (BRCv) within the CTE is necessary for localization to sites of MMC-induced damage

  • Create domain deletion mutants and assess functional impacts on each repair pathway

Research findings indicate that MCM9 has distinct mechanistic roles: in HR, it primarily functions with MCM8 to promote DNA synthesis during repair, while in MMR, it acts as a helicase to unwind mismatched DNA strands and recruits MLH1 to chromatin .

How does the HROB-MCM8-MCM9 pathway contribute to genomic stability and what methodologies can assess this function?

The HROB-MCM8-MCM9 pathway plays a crucial role in maintaining genomic stability through promoting homologous recombination. To investigate this pathway:

• Synthetic lethality screening:

  • CRISPR/Cas9 screens reveal that HROB and MCM8-MCM9 act in the same pathway for cisplatin resistance

  • HROB and HELQ act in parallel pathways, with double knockouts showing severely impaired HR

  • Design an ELISA-based screen using MCM9 Antibody, HRP conjugated to identify additional pathway components

• Epistasis analysis methodologies:

  • Generate single and double knockouts of pathway components

  • Measure sensitivity to DNA damaging agents quantitatively

  • Assess HR efficiency using reporter assays

  • Quantify chromosomal aberrations following damage

• In vitro biochemical reconstitution:

  • Purify recombinant HROB, MCM8, and MCM9 proteins

  • Assess helicase activity on model DNA substrates

  • Determine how HROB influences MCM8-MCM9 helicase activity

The MCM8-MCM9 complex forms a helicase related to the MCM2-MCM7 replicative helicase but plays a specialized role in HR . Evidence suggests that HROB may function as a helicase loader for MCM8-MCM9, similar to how gp59 acts as a helicase loader during bacteriophage T4 recombination-dependent DNA replication .

What are the best experimental designs to study the C-terminal domain of MCM9 in protein localization and function?

The C-terminal extension (CTE) of MCM9 comprises 42% of the total protein length and contains critical functional motifs despite being largely disordered . To study this domain:

• Motif-specific mutational analysis:

  • Target the bipartite-like nuclear localization signal (NLS) in the CTE required for nuclear import of both MCM8 and MCM9

  • Mutate the BRC variant (BRCv) motif necessary for localization to mitomycin C-induced damage sites

  • Generate stable cell lines expressing these mutants and assess localization and function

• Fluorescence recovery after photobleaching (FRAP):

  • Create fluorescently tagged wild-type and CTE-mutant MCM9 constructs

  • Analyze recruitment dynamics to laser-induced DNA damage

  • Quantify mobility and retention at damage sites

• Domain swapping experiments:

  • Replace the MCM9 CTE with heterologous domains having similar predicted functions

  • Test whether these chimeric proteins retain ability to:

    • Localize to the nucleus

    • Recruit to damage sites

    • Interact with MCM8 and other repair factors

    • Complement MCM9 deficiency

Research indicates that the CTE contains two unique motifs critical for function: an unconventional "bipartite-like" NLS consisting of two positively charged amino acid stretches separated by a long intervening sequence, and a variant BRC motif similar to that found in other HR helicases .

What are common technical challenges when working with MCM9 Antibody, HRP conjugated and how can they be addressed?

When working with MCM9 Antibody, HRP conjugated, researchers may encounter these challenges:

• High background signal in ELISA applications:

  • Optimize blocking conditions (try different blockers like BSA, milk, commercial blockers)

  • Increase washing stringency (add 0.1% Tween-20 to wash buffers)

  • Titrate antibody concentration to find optimal signal-to-noise ratio

  • Pre-absorb antibody with cell lysate from MCM9-knockout cells

• Weak or variable signal detection:

  • Ensure target accessibility by optimizing sample preparation

  • For nuclear proteins like MCM9, use nuclear extraction protocols with multiple detergents

  • Enhance detection with amplification systems like tyramide signal amplification

  • Consider chromatin extraction protocols to enrich for DNA-bound MCM9 fraction

• Cross-reactivity with related MCM family proteins:

  • Validate specificity using MCM9-knockout controls

  • Perform peptide competition assays with the immunizing peptide (1-391AA)

  • Compare results with alternative MCM9 antibodies targeting different epitopes

• Detecting damage-induced MCM9 localization changes:

  • Use appropriate damage induction (mitomycin C specifically induces MCM9 localization)

  • Allow sufficient time for recruitment (optimize time-course experiments)

  • Consider subcellular fractionation approaches rather than whole-cell lysates

How can researchers quantitatively assess MCM9 recruitment to sites of DNA damage?

For quantitative analysis of MCM9 recruitment to DNA damage sites:

• Chromatin immunoprecipitation (ChIP) coupled with qPCR:

  • Induce site-specific DNA damage (e.g., with endonucleases)

  • Perform ChIP using MCM9 antibody

  • Quantify enrichment at damage sites versus control regions by qPCR

  • Use the HRP-conjugated antibody in downstream ELISA validation

• High-content imaging approaches:

  • Induce localized DNA damage (laser microirradiation or localized chemical treatment)

  • Detect MCM9 with primary antibody followed by fluorescent secondary

  • Quantify recruitment kinetics through automated image analysis

  • Normalize to damage markers (γH2AX, 53BP1)

• Proximity ligation assay (PLA) quantification:

  • Combine antibodies against MCM9 and damage markers

  • Quantify PLA signals per nucleus as measure of recruitment

  • Compare kinetics in different genetic backgrounds

Research indicates that MCM9 recruitment to damage sites depends on the MRN complex and occurs downstream of BRCA2 and RAD51 . This recruitment is critical for processing repair intermediates and facilitating homologous recombination.

How can MCM9 Antibody, HRP conjugated be used in cancer research studies?

For cancer research applications of MCM9 Antibody, HRP conjugated:

• Analysis of homologous recombination deficiency (HRD):

  • Quantify MCM9 expression and localization in HRD versus HR-proficient tumors

  • Correlate with response to PARP inhibitors or platinum-based chemotherapies

  • Use the HRP-conjugated antibody in tissue microarray ELISA applications

• Biomarker development methodologies:

  • Analyze MCM9 recruitment to damage sites as a functional HRD assay

  • Develop ELISA-based screening for MCM9 pathway defects in tumor samples

  • Correlate MCM9 pathway function with genomic instability signatures

• Synthetic lethality studies:

  • CRISPR screens identified MCM9 as synthetically lethal with ATR and PARP inhibition

  • Measure MCM9 expression/function in tumors to predict therapy response

  • Use MCM9 status to stratify patients for clinical trials

Research indicates that genes involved in promoting cellular resistance to both ATR and PARP inhibitors are enriched for HR factors, including MCM9 . This suggests that MCM9 expression or function could serve as a biomarker for predicting response to these targeted therapies.

What methodological approaches can assess MCM9's role in gametogenesis and fertility research?

To investigate MCM9's functions in gametogenesis and fertility:

• Analysis of meiotic progression:

  • HROB knockout mice show infertility due to germ cell depletion and meiotic arrest in prophase I

  • The MCM8-MCM9 complex plays a key role during gametogenesis

  • Use immunohistochemistry with MCM9 antibodies to assess expression patterns during meiotic stages

• Meiotic recombination assessment:

  • Analyze crossover formation and distribution in MCM9-deficient models

  • Quantify double-strand break repair efficiency during meiotic prophase

  • Correlate with fertility outcomes

• in vitro fertilization studies:

  • Assess MCM9 expression in gametes using HRP-conjugated antibody in ELISA format

  • Correlate expression levels with fertilization success and embryo development

  • Develop potential diagnostic tools for infertility evaluation

The MCM8-MCM9 complex's role in homologous recombination appears particularly critical during gametogenesis, as evidenced by the infertility phenotypes observed in knockout models . This suggests MCM9 detection could have applications in reproductive medicine research.