MCM7 PAT1G8AT Antibody

What is MCM7 and why is it significant in biological research?

MCM7 is a highly conserved mini-chromosome maintenance protein vital for eukaryotic genome replication initiation. It forms part of a hexameric protein complex (the MCM complex) that serves as a key component of the pre-replication complex (pre-RC), which is crucial for replication fork formation and recruitment of DNA replication-related proteins. The MCM complex, comprised of MCM2, MCM4, MCM6, and MCM7 proteins, possesses DNA helicase activity that enables DNA unwinding during replication. MCM7 is particularly significant in research because it provides insights into fundamental cellular processes of DNA replication, cell cycle regulation, and genomic stability. Understanding MCM7 function contributes to our knowledge of cancer development mechanisms, as dysregulation of DNA replication is a hallmark of many cancers .

What are the key specifications of the MCM7 PAT1G8AT Antibody?

The MCM7 PAT1G8AT antibody is a mouse monoclonal antibody derived from hybridization of mouse F0 myeloma cells with spleen cells from BALB/c mice immunized with recombinant human MCM7 protein (amino acids 1-414) purified from E. coli. This antibody belongs to the IgG2b isotype with kappa light chains and has been purified using protein-G affinity chromatography. It is typically formulated at 1mg/ml concentration in PBS (pH 7.4) with 0.1% Sodium Azide as a preservative. The antibody has been validated for applications including ELISA and Western blot analysis, with a molecular weight detection of approximately 80 kDa, corresponding to the MCM7 protein .

What species reactivity has been confirmed for this antibody?

Based on available information, the MCM7 PAT1G8AT antibody and related MCM7 antibodies have demonstrated reactivity across multiple species. While the specific PAT1G8AT clone reactivity isn't explicitly detailed in all provided information, similar MCM7 antibodies have shown reactivity to human (H), mouse (M), rat (R), hamster (Hm), monkey (Mk), and dog (Dg) samples. Western blot analysis and immunofluorescent data for some MCM7 antibodies indicate stronger reactivity to primate proteins compared to rodent proteins. This cross-species reactivity makes the antibody valuable for comparative studies across different model organisms .

What applications has the MCM7 PAT1G8AT Antibody been validated for?

• Western Blotting (WB): Typically used at 1:1000 dilution

• Immunoprecipitation (IP): Used at 1:100 dilution

• Immunohistochemistry (IHC) on paraffin-embedded samples: Used at 1:200 to 1:800 dilution

• Immunofluorescence (IF)/Immunocytochemistry: Used at 1:100 to 1:200 dilution

For optimal results with each application, it is recommended to titrate the antibody concentration for each specific experimental setup and sample type .

How should I design a Western blot experiment using MCM7 PAT1G8AT Antibody?

When designing a Western blot experiment using the MCM7 PAT1G8AT antibody, consider the following protocol:

• Sample preparation:

  • Extract total protein from cells or tissue using a suitable lysis buffer containing protease inhibitors

  • Quantify protein concentration using a reliable method (Bradford, BCA, etc.)

  • Use 20-50 μg of total protein per lane

• Gel electrophoresis:

  • Use an 8-10% SDS-PAGE gel (MCM7 has a molecular weight of approximately 80 kDa)

  • Include appropriate molecular weight markers

• Transfer:

  • Transfer proteins to a PVDF or nitrocellulose membrane

  • Verify transfer efficiency with reversible staining (Ponceau S)

• Blocking:

  • Block membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute MCM7 PAT1G8AT antibody 1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

• Washing:

  • Wash membrane 3-4 times with TBST, 5 minutes each

• Secondary antibody incubation:

  • Use an appropriate anti-mouse IgG HRP-conjugated secondary antibody

  • Dilute according to manufacturer's recommendation

  • Incubate for 1 hour at room temperature

• Detection:

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Expose to X-ray film or use a digital imaging system

• Analysis:

  • The expected band for MCM7 should be observed at approximately 80 kDa

  • Include positive controls (cell lines known to express MCM7)

  • Use loading controls (β-actin, GAPDH) to normalize expression levels

What are the critical parameters for optimizing immunofluorescence with this antibody?

When optimizing immunofluorescence using the MCM7 PAT1G8AT antibody, several critical parameters should be considered:

• Fixation method:

  • For nuclear proteins like MCM7, 4% paraformaldehyde for 10-15 minutes is usually effective

  • Some epitopes may require methanol fixation (-20°C for 10 minutes)

  • Test both methods to determine optimal epitope preservation

• Permeabilization:

  • Use 0.1-0.5% Triton X-100 in PBS for 10 minutes

  • For nuclear proteins, ensure adequate permeabilization to allow antibody access

• Blocking:

  • Block with 1-5% BSA or normal serum (from the species of secondary antibody) in PBS for 30-60 minutes

  • Include 0.1% Triton X-100 in blocking buffer to maintain permeabilization

• Antibody concentration:

  • Start with a 1:100 to 1:200 dilution of MCM7 PAT1G8AT antibody

  • Perform a dilution series to identify optimal signal-to-noise ratio

• Incubation conditions:

  • Incubate primary antibody overnight at 4°C or for 1-2 hours at room temperature

  • Incubate secondary antibody for 1 hour at room temperature in the dark

• Controls:

  • Include a negative control (omitting primary antibody)

  • Use a cell line with known MCM7 expression patterns as a positive control

  • Consider peptide competition assay to confirm specificity

• Counterstaining:

  • Use DAPI or Hoechst for nuclear counterstaining

  • Since MCM7 is nuclear, this provides contextual localization information

What cell lines serve as appropriate positive controls for MCM7 antibody validation?

While the search results don't explicitly mention specific cell lines as positive controls for the MCM7 PAT1G8AT antibody, MCM7 is broadly expressed in proliferating cells. Based on general knowledge of MCM7 expression patterns, the following can be recommended as positive controls:

• Cell lines:

  • HeLa cells (human cervical cancer)

  • MCF7 cells (human breast cancer)

  • HEK293 cells (human embryonic kidney)

  • A549 cells (human lung carcinoma)

  • NIH/3T3 cells (mouse fibroblasts)

• Tissue samples:

  • Proliferating epithelial tissues

  • Embryonic tissues with high proliferation rates

  • Germinal centers of lymphoid tissues

  • Intestinal crypts

  • Hair follicles in anagen phase

When using these positive controls, it's important to note that MCM7 expression typically correlates with cell proliferation status. Actively dividing cell populations will show stronger MCM7 expression. Since MCM7 is involved in DNA replication initiation, its expression is cell cycle-dependent, with highest levels during late G1 and S phases .

What are common causes of high background when using this antibody in immunohistochemistry?

High background in immunohistochemistry with MCM7 PAT1G8AT antibody can result from several factors:

• Insufficient blocking:

  • Extend blocking time to 1-2 hours

  • Increase blocking agent concentration (5-10% normal serum or BSA)

  • Add 0.1-0.3% Triton X-100 to blocking buffer to reduce non-specific binding

• Antibody concentration too high:

  • Perform a titration series (e.g., 1:100, 1:200, 1:400, 1:800)

  • Optimal dilution provides specific signal with minimal background

• Cross-reactivity issues:

  • Use species-specific blocking agents that match secondary antibody source

  • Pre-absorb primary antibody with tissue powder from the species being tested

  • Consider using monovalent Fab fragments to block endogenous immunoglobulins

• Endogenous peroxidase activity:

  • Block with 0.3-3% hydrogen peroxide in methanol for 10-30 minutes before antibody incubation

  • For fluorescence-based detection, use Sudan Black B (0.1-0.3%) to reduce autofluorescence

• Inadequate washing:

  • Increase washing duration and number of wash steps

  • Use gentle agitation during washing

  • Add 0.05-0.1% Tween-20 to wash buffers

What should I do if I observe no signal in Western blot when using this antibody?

If you observe no signal in Western blot using the MCM7 PAT1G8AT antibody, consider the following troubleshooting steps:

• Verify protein expression and loading:

  • Confirm protein loading with Ponceau S staining or total protein stains

  • Ensure adequate protein amount (30-50 μg total protein per lane)

  • Use a positive control cell line known to express MCM7

  • Check expression of housekeeping proteins (β-actin, GAPDH) as loading controls

• Antibody-related issues:

  • Verify antibody integrity (avoid repeated freeze-thaw cycles)

  • Increase antibody concentration (try 1:500 or 1:250 dilution)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Check secondary antibody reactivity with a different primary antibody

• Protein detection issues:

  • Ensure ECL substrate is fresh and properly mixed

  • Increase film exposure time or detector sensitivity

  • Use a more sensitive detection system (enhanced ECL or fluorescent detection)

• Protein transfer problems:

  • Verify transfer efficiency with reversible protein staining

  • Adjust transfer conditions for high molecular weight proteins (80 kDa)

  • Use PVDF membrane which may bind proteins more efficiently than nitrocellulose

  • Consider wet transfer instead of semi-dry transfer for better efficiency

• Epitope accessibility:

  • The epitope might be masked or destroyed during processing

  • Adjust SDS concentration in sample buffer

  • Consider native vs. reducing conditions

How can I address cross-reactivity issues with this antibody?

To address potential cross-reactivity issues with the MCM7 PAT1G8AT antibody:

• Antibody validation:

  • Perform peptide competition assays using the immunizing peptide

  • Test the antibody in MCM7 knockout or knockdown samples as negative controls

  • Compare staining patterns with other validated MCM7 antibodies recognizing different epitopes

• Selectivity optimization:

  • Increase antibody dilution to reduce non-specific binding (start with 1:2000 or higher)

  • Add 0.1-0.5% non-ionic detergent (Triton X-100 or Tween-20) to antibody diluent

  • Include 0.1-0.5M NaCl in antibody diluent to reduce ionic interactions

  • Add 1-5% non-fat dry milk or BSA to antibody diluent

• Pre-adsorption techniques:

  • Pre-incubate the antibody with tissue/cell lysate from species where cross-reactivity occurs

  • Use commercial pre-adsorption kits to remove cross-reactive antibodies

• Data analysis approaches:

  • Always include appropriate negative controls

  • Verify band identity using siRNA knockdown or CRISPR knockout models

  • For Western blots showing multiple bands, perform subcellular fractionation to identify specific localizations

• Technical considerations:

  • When studying MCM family proteins, be aware of sequence homology that might cause cross-reactivity

  • The MCM2-7 complex members share structural similarities that may affect antibody specificity

  • Consider using antibodies targeting unique regions of MCM7 rather than conserved domains

How can this antibody be used to study cell cycle progression?

The MCM7 PAT1G8AT antibody can be valuable for studying cell cycle progression through several experimental approaches:

• Immunofluorescence-based cell cycle analysis:

  • Combine MCM7 staining with DNA content analysis (DAPI or propidium iodide)

  • Co-stain with cell cycle markers (e.g., cyclins, phospho-histone H3)

  • MCM7 shows characteristic patterns during different cell cycle phases:

    • G1 phase: MCM7 begins loading onto chromatin

    • S phase: MCM7 is maximally loaded but begins to dissociate as replication proceeds

    • G2/M phases: MCM7 is largely displaced from chromatin

• Chromatin association dynamics:

  • Perform biochemical fractionation to separate chromatin-bound from soluble MCM7

  • Track changes in chromatin-bound MCM7 during synchronized cell cycle progression

  • Analyze kinetics of MCM7 loading/unloading at specific genomic loci using ChIP

• Cell synchronization experiments:

  • Synchronize cells at different cell cycle phases (double thymidine block, nocodazole, etc.)

  • Monitor MCM7 levels and localization as cells progress through the cycle

  • Correlate MCM7 dynamics with DNA replication timing

• Proliferation marker studies:

  • Use MCM7 as a proliferation marker in tissues or cell cultures

  • Compare with established proliferation markers (Ki-67, PCNA)

  • MCM7 may identify replication-licensed cells before they enter S phase

What experimental approaches can be used to study MCM7's role in DNA replication?

To investigate MCM7's role in DNA replication using the PAT1G8AT antibody, consider these experimental approaches:

• Chromatin immunoprecipitation (ChIP):

  • Perform ChIP with MCM7 PAT1G8AT antibody to identify genomic binding sites

  • Combine with sequencing (ChIP-seq) to map genome-wide MCM7 binding

  • Compare MCM7 binding sites with known replication origins

  • Analyze temporal dynamics of MCM7 loading at specific origins during S phase

• DNA replication assays:

  • Use DNA fiber analysis to measure replication fork progression

  • Combine with MCM7 immunodepletion or knockdown to assess functional impact

  • Analyze effects of MCM7 mutations on replication fork speed and stability

  • Perform DNA combing to visualize active replication origins and fork symmetry

• Protein-protein interaction studies:

  • Immunoprecipitate MCM7 to identify associated proteins in the pre-replication complex

  • Perform mass spectrometry analysis of co-precipitated proteins

  • Use proximity ligation assays to visualize MCM7 interactions with other replication factors

  • Analyze how these interactions change during origin licensing and activation

• Replication stress response:

  • Study MCM7 dynamics following treatment with replication stress inducers (hydroxyurea, aphidicolin)

  • Analyze MCM7 post-translational modifications in response to replication stress

  • Examine recruitment of checkpoint proteins to MCM7-containing complexes

• Origin licensing and activation:

  • Combine MCM7 staining with nascent DNA labeling (EdU incorporation)

  • Correlate MCM7 chromatin binding with replication timing domains

  • Study interactions between MCM7 and origin activation factors (CDC45, GINS)

How can this antibody be applied in studies of cancer cell proliferation?

The MCM7 PAT1G8AT antibody offers several approaches for studying cancer cell proliferation:

• Proliferation marker analysis:

  • Compare MCM7 expression levels between normal and cancer cells

  • Use tissue microarrays to assess MCM7 as a proliferation marker across tumor types

  • Correlate MCM7 expression with established proliferation markers (Ki-67, PCNA)

  • MCM7 may identify cells licensed for replication but not yet actively dividing

• Prognostic biomarker studies:

  • Evaluate MCM7 expression in tumor samples with known clinical outcomes

  • Perform multivariate analysis to assess MCM7's independent prognostic value

  • Compare MCM7 with established clinical parameters for risk stratification

  • Analyze correlation between MCM7 levels and tumor grade/stage

• Therapeutic response monitoring:

  • Track changes in MCM7 expression following anti-proliferative treatments

  • Use MCM7 as a pharmacodynamic marker for drugs targeting cell cycle machinery

  • Compare MCM7 dynamics with tumor growth inhibition in preclinical models

  • Study replication stress-induced changes in MCM7 following chemotherapy

• Multi-parameter analysis:

  • Perform multiplexed immunofluorescence to study MCM7 with other proliferation markers

  • Combine with cell cycle markers to distinguish G1, S, G2, and M populations

  • Correlate with DNA damage markers to identify replication stress

  • Use image cytometry for single-cell quantification in heterogeneous tumors

How should I quantify and interpret MCM7 expression data across experimental contexts?

Quantification and interpretation of MCM7 expression data requires careful consideration of several factors depending on the experimental context:

• Western blot quantification:

  • Normalize MCM7 band intensity to loading controls (β-actin, GAPDH, total protein)

  • Use digital imaging and analysis software for density quantification

  • Perform replicate experiments (n≥3) for statistical validation

  • Present data as fold change relative to control conditions

  • Consider semi-quantitative analysis for comparing expression across cell lines or tissue types

• Immunohistochemistry (IHC) scoring:

  • Develop a consistent scoring system (e.g., H-score, combining intensity and percentage)

  • Score MCM7 staining as:

    • Percentage of positive cells (0-100%)

    • Staining intensity (0=negative, 1=weak, 2=moderate, 3=strong)

    • H-score = Σ (percentage × intensity) ranging from 0-300

  • Consider automated image analysis for objective quantification

  • Use pattern recognition to distinguish nuclear vs. cytoplasmic staining

  • Compare with adjacent normal tissue as internal control

• Immunofluorescence analysis:

  • Measure nuclear fluorescence intensity using appropriate image analysis software

  • Subtract background signal from non-specific staining

  • Normalize to nuclear area or DNA content (DAPI)

  • Consider 3D confocal analysis for volume-based quantification

  • Analyze co-localization with other proteins of interest using correlation coefficients

• Interpretation considerations:

  • Cell cycle dependency: MCM7 expression and localization change throughout the cell cycle

  • Proliferation status: Compare MCM7 levels between quiescent and proliferating populations

  • Subcellular localization: Distinguish between chromatin-bound and soluble fractions

  • Post-translational modifications: Consider how modifications affect antibody recognition

What are common pitfalls in data interpretation when studying MCM7?

When studying MCM7 using antibody-based methods, researchers should be aware of these common pitfalls in data interpretation:

• Cell cycle dependency misinterpretation:

  • MCM7 expression and localization fluctuate during cell cycle progression

  • Changes may reflect cell cycle distribution shifts rather than true expression differences

  • Solution: Use cell cycle markers in parallel or synchronize cells when comparing conditions

• Cross-reactivity with other MCM proteins:

  • MCM family proteins share sequence homology

  • Antibodies may recognize multiple MCM proteins despite claimed specificity

  • Solution: Validate antibody specificity using knockdown/knockout controls or mass spectrometry

• Confounding of proliferation vs. expression changes:

  • Changes in apparent MCM7 levels may simply reflect altered proliferation rates

  • Solution: Normalize to other proliferation markers or cell cycle analyses

• Epitope masking effects:

  • Post-translational modifications or protein-protein interactions may mask epitopes

  • Different fixation methods may variably expose epitopes

  • Solution: Use multiple antibodies targeting different epitopes or validate under specific conditions

• Chromatin association misconceptions:

  • Total MCM7 levels don't necessarily reflect functionally relevant chromatin-bound fractions

  • Solution: Perform biochemical fractionation to distinguish chromatin-bound from soluble pools

• Signal quantification errors:

  • Saturation of signal in highly expressing samples can compress dynamic range

  • Non-linear relationship between protein amount and signal intensity

  • Solution: Perform dilution series to ensure measurements fall within linear range

How can I integrate MCM7 expression data with other DNA replication markers?

Integrating MCM7 expression data with other DNA replication markers provides a more comprehensive understanding of replication pathways. Here's how to approach this integration:

• Multi-protein analysis strategies:

  • Design antibody panels targeting different components of the DNA replication machinery:

    • Pre-replication complex (ORC1-6, CDC6, CDT1, MCM2-7)

    • Pre-initiation complex (CDC45, GINS complex, MCM10)

    • Replisome components (DNA polymerases, PCNA, RFC)

    • Regulatory factors (Geminin, Cyclin/CDKs, DDK)

  • Use multiplexed immunofluorescence or sequential immunohistochemistry

  • Perform parallel Western blots from the same samples

  • Consider reverse phase protein arrays for high-throughput analysis

• Functional correlation approaches:

  • Combine MCM7 detection with nascent DNA synthesis markers:

    • EdU or BrdU incorporation for active replication

    • PCNA patterns for replication factory visualization

    • γH2AX foci for replication stress

  • Analyze temporal relationships between MCM7 loading and replication initiation

  • Correlate MCM7 levels with replication timing domains

• Pathway visualization and analysis:

  • Create heat maps or correlation matrices of replication protein expression

  • Use principal component analysis to identify patterns across multiple markers

  • Implement pathway analysis software to visualize interactions

  • Develop network models incorporating protein interactions and regulatory relationships

• Temporal dynamics analysis:

  • Perform time-course experiments following cell cycle synchronization

  • Track sequential recruitment of replication factors

  • Analyze kinetics of assembly and disassembly

  • Develop mathematical models of replication dynamics