DMC1 Antibody

What types of DMC1 antibodies are available for research?

Several types of DMC1 antibodies are currently available for research applications:

• Mouse monoclonal antibodies (such as A-6) that detect DMC1 protein of mouse, rat, and human origin

• Rabbit polyclonal antibodies designed against human DMC1 protein fragments

These antibodies come in various forms:

• Non-conjugated forms for general applications

• Conjugated forms including:

  • Agarose conjugates for immunoprecipitation

  • Horseradish peroxidase (HRP) conjugates for enhanced detection

  • Phycoerythrin (PE) conjugates for flow cytometry

  • Fluorescein isothiocyanate (FITC) conjugates for fluorescence applications

  • Multiple Alexa Fluor® conjugates for advanced fluorescence imaging

The selection of the appropriate antibody depends on the experimental technique, species of interest, and specific research objectives.

What are the validated applications for DMC1 antibodies?

DMC1 antibodies have been validated for multiple experimental applications:

For western blotting, DMC1 antibodies have been successfully used with various cell lysates including Raji (human Burkitt's lymphoma) and K562 (human chronic myelogenous leukemia) cell lines, with a predicted band size of 38 kDa . For immunohistochemistry, positive staining has been demonstrated in human testis tissue , consistent with DMC1's known role in meiotic recombination.

How should DMC1 antibodies be stored and handled?

While the search results don't provide specific storage information for DMC1 antibodies, general best practices for antibody storage and handling should be followed:

• Store antibodies according to manufacturer's recommendations, typically at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles by aliquoting the antibody upon receipt

• For working dilutions, store at 4°C for short-term use (1-2 weeks)

• Protect conjugated antibodies (especially fluorophore-conjugated) from light exposure

• When diluting antibodies, use appropriate buffers as recommended by the manufacturer

• Record lot numbers and validate each new lot against previous lots for consistency

These practices will help maintain antibody performance and extend shelf-life for research applications.

What are the optimal conditions for immunoprecipitation with DMC1 antibodies?

For effective immunoprecipitation with DMC1 antibodies, researchers should consider the following protocol based on published research:

• Binding conditions: For anti-FLAG immunoprecipitations, mix 2 μg of purified recombinase (yDmc1, yRad51, or hDmc1) with 1 μg MLH complex (yMlh1-Mlh3 or yMlh1-Pms1) in 100–400 μl total volume Binding/washing buffer at 4°C for 30 minutes .

• DNase treatment (if required): Add 2 units of DNase I along with MgCl₂ (final concentration 2.5 mM). Incubate DNase I treated samples at 37°C for 5 minutes before proceeding with the protein mixture incubation at 4°C for 30 minutes .

• Quality control: To verify DNA degradation when using DNase, remove 5 μl of DNase treated samples after 37°C incubation, treat with Proteinase K, and incubate at room temperature for 15 minutes before analyzing on a 1% agarose gel .

• Detection methodology: For immunoprecipitation analyses, proteins should be separated by SDS-PAGE and transferred to a 0.2μm PVDF membrane. Transfer conditions can vary: 100V for 2 hrs, 70V for 4 hrs, or 30V overnight .

When working with meiotic cell extracts, physiological salt concentrations (150 mM NaCl) should be maintained to preserve native protein interactions .

How can I optimize western blotting protocols for DMC1 detection?

Optimizing western blotting for DMC1 detection requires attention to several key parameters:

• Sample preparation:

  • For cell lines: Prepare whole cell lysates from relevant cell types (e.g., Raji or K562 cells have shown good expression)

  • For tissue samples: Testis tissue is particularly relevant due to high DMC1 expression

• Antibody dilutions:

  • Primary antibody: Use rabbit polyclonal DMC1 antibody at 1/500 dilution for cell lysates

  • Secondary antibody: Use appropriate species-specific secondary antibody (e.g., goat polyclonal to rabbit IgG) at 1/50000 dilution

• Expected results:

  • Predicted band size: 38 kDa

  • Validate specificity with positive and negative controls

• Transfer conditions:

  • Transfer proteins to a 0.2μm PVDF membrane

  • Transfer at either 100V for 2 hrs, 70V for 4 hrs, or 30V overnight

• Detection system:

  • Use enhanced chemiluminescence (ECL) substrates for sensitive detection

  • Image using either film exposure or digital imaging systems (e.g., ChemiDoc MP)

  • Quantify band intensity using appropriate software (e.g., Image Lab)

For multiplexing, DMC1 detection can be combined with housekeeping controls such as glucose-6-phosphate dehydrogenase (G6PDH), which can be detected with rabbit anti-G6PDH (1:10,000) and peroxidase conjugated anti-rabbit (1:20,000) .

What are the recommended protocols for immunofluorescence using DMC1 antibodies?

For immunofluorescence detection of DMC1, the following protocol is recommended based on published literature:

• Cell preparation:

  • Fixed cell lines: HeLa cells have been successfully used for DMC1 immunostaining

  • Meiotic cells: Prepare from appropriate tissues (e.g., testis)

• Antibody dilutions:

  • Primary antibody: Use DMC1 antibody at 1/100 dilution

  • Secondary antibody: Use appropriate fluorophore-conjugated secondary antibody

• Controls:

  • Include negative controls (secondary antibody only) to assess background

  • Consider positive controls with known DMC1 expression patterns

• Counterstaining:

  • Nuclear counterstain (e.g., DAPI at 0.1 mg/ml) helps visualize nuclear localization

  • For meiotic studies, consider co-staining with synaptonemal complex proteins

• Imaging:

  • Use appropriate filters for the selected fluorophore

  • Capture multiple fields to ensure representative imaging

  • For meiotic studies, Z-stack imaging may be necessary to capture the full nuclear volume

When working with tissues, paraffin-embedded human testis tissue has shown positive staining for DMC1 using antibody at 1/100 dilution in immunohistochemical analysis .

How can I verify the specificity of DMC1 antibody staining?

Verifying antibody specificity is crucial for reliable research outcomes. For DMC1 antibodies, consider the following approaches:

• Positive and negative tissue controls:

  • Positive control: Use testis tissue which has high DMC1 expression

  • Negative control: Use somatic tissues where DMC1 should not be expressed

• Cellular expression pattern validation:

  • DMC1 should show nuclear localization, particularly during meiotic prophase

  • Expression should be restricted to germ cells and absent in somatic cells

• Knockdown/knockout validation:

  • Use siRNA/shRNA knockdown or CRISPR/Cas9 knockout systems

  • Compare staining intensity between control and DMC1-depleted samples

• Multiple antibody validation:

  • Use antibodies from different sources or raised against different epitopes

  • Consistent staining patterns across antibodies support specificity

• Western blot correlation:

  • Confirm that tissues/cells showing positive immunostaining also show the expected 38 kDa band in western blotting

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide

  • Specific staining should be blocked by peptide competition

When publishing research using DMC1 antibodies, thorough validation data should be included to establish confidence in the results.

How can DMC1 antibodies be used to study protein-protein interactions in meiotic recombination?

DMC1 antibodies can be powerful tools for investigating protein-protein interactions in meiotic recombination through several approaches:

• Co-immunoprecipitation (Co-IP):

  • Use DMC1 antibodies to pull down DMC1 and its interacting partners

  • Research has successfully used this approach to identify interactions between yeast Mlh1-Mlh3 and DMC1

  • Protocol example: For anti-FLAG immunoprecipitations, mix 2 μg of purified recombinase (yDmc1, yRad51, or hDmc1) with 1 μg MLH complex (yMlh1-Mlh3 or yMlh1-Pms1) in binding buffer at 4°C

• Testing DNA-dependence of interactions:

  • Add DNase I (2 units) with MgCl₂ (2.5 mM final) and incubate at 37°C for 5 minutes

  • The DMC1-Mlh3 interaction was retained in the presence of DNase I and in the absence of ATP, suggesting a direct protein-protein interaction rather than DNA-mediated association

• Temporal studies during meiosis:

  • Extract samples at defined timepoints after meiotic induction

  • DMC1-Mlh3 interaction has been detected at 6 and 8 hours post-meiotic induction, corresponding to meiotic prophase

• Cross-species interaction studies:

  • Test conservation of interactions across species

  • Yeast Mlh1-Mlh3 robustly immunoprecipitated yeast DMC1 and yeast Rad51 but not human DMC1, indicating species-specific interaction surfaces

These approaches can reveal critical insights into the molecular mechanisms of meiotic recombination and the role of DMC1 in this process.

What techniques can be combined with DMC1 immunostaining to study meiotic progression?

Researchers can combine DMC1 immunostaining with complementary techniques to gain comprehensive insights into meiotic progression:

• DAPI staining for meiotic staging:

  • Stain nuclei with DAPI (0.1 mg/ml)

  • Track progression by counting cells with 1, 2, 3/4 nuclei

  • Analyze at least 600 cells per genotype per timepoint (except 0-hr timepoint where 300 cells are sufficient)

• Co-immunostaining with stage-specific markers:

  • Synaptonemal complex proteins (e.g., SYCP3, SYCP1)

  • Phosphorylated histone H2AX (γH2AX) for DNA damage sites

  • MLH1/MLH3 for crossover sites

  • RAD51 for recombination intermediates

• Chromosome spreads:

  • Prepare chromosome spreads from meiotic cells

  • Perform immunofluorescence on spreads for higher resolution imaging of chromosomal structures

  • Allows visualization of DMC1 foci along chromosomes

• Time-course analysis:

  • Collect samples at various timepoints after meiotic induction (0, 4, 6, 8 hours)

  • Monitor changes in protein expression, localization, and interactions

  • Consider culture volume effects on meiotic progression (larger culture volumes may show 2-hour delays)

• Genetic manipulation:

  • Use β-estradiol-inducible systems (10 nM for complementation, 1 μM for overexpression)

  • Study effects of mutations or overexpression on DMC1 localization and function

By combining these approaches, researchers can correlate DMC1 dynamics with specific stages of meiotic progression and understand how perturbations affect meiotic recombination outcomes.

How do DMC1 antibodies help differentiate between mitotic and meiotic recombination mechanisms?

DMC1 antibodies are valuable tools for distinguishing meiotic from mitotic recombination mechanisms due to the specific expression and function of DMC1 in meiosis:

• Cell type-specific expression:

  • DMC1 is predominantly expressed in meiotic cells and is absent in mitotic cells

  • Immunostaining with DMC1 antibodies can effectively identify cells undergoing meiotic recombination

  • Positive staining in human testis tissue confirms meiosis-specific expression

• Co-localization studies:

  • In mitotic cells, only RAD51 foci are observed at sites of DNA damage

  • In meiotic cells, both DMC1 and RAD51 co-localize at early recombination nodules

  • DMC1 antibodies allow visualization of this distinctive meiotic pattern

• Protein interaction networks:

  • DMC1 interacts with meiosis-specific factors like Mlh3

  • Co-immunoprecipitation using DMC1 antibodies can pull down these meiosis-specific interactors

  • The yeast Mlh1-Mlh3 complex robustly immunoprecipitates yeast DMC1, highlighting meiosis-specific protein interactions

• Temporal dynamics:

  • DMC1 shows distinct temporal patterns during meiotic prophase

  • DMC1-Mlh3 interaction is detected at specific timepoints (6 and 8 hours post-induction)

  • This temporal regulation differs from mitotic recombination mechanisms

Understanding these differences is critical for research on reproductive biology, fertility disorders, and evolutionary mechanisms of genetic recombination.

What are the current challenges in using DMC1 antibodies for studying meiotic recombination intermediates?

Despite their utility, researchers face several challenges when using DMC1 antibodies to study meiotic recombination:

• Specificity concerns:

  • Cross-reactivity with RAD51 due to high sequence homology (~50% identity)

  • Need for careful validation using appropriate controls

  • Potential for non-specific nuclear staining in some tissue preparations

• Technical limitations:

  • Variable antibody performance across different experimental conditions

  • Challenges in preserving nuclear architecture during fixation procedures

  • Limited availability of antibodies recognizing specific post-translational modifications of DMC1

• Species-specific considerations:

  • Species differences in DMC1 structure affect antibody compatibility

  • Yeast Mlh1-Mlh3 immunoprecipitates yeast DMC1 but not human DMC1, indicating species-specific epitopes

  • Need for species-validated antibodies for comparative studies

• Temporal dynamics complexity:

  • Meiotic cultures show variability in synchronization

  • Large culture volumes can delay meiotic progression by ~2 hours compared to smaller volumes

  • Requires careful experimental design and appropriate controls

• Quantification challenges:

  • Difficulty in standardizing DMC1 foci counting

  • Variability in staining intensity across different preparations

  • Need for automated analysis tools for consistent quantification

Addressing these challenges requires rigorous controls, method standardization, and careful interpretation of results when working with DMC1 antibodies.

Why might western blotting for DMC1 show unexpected band patterns?

When western blotting for DMC1 produces unexpected band patterns, consider the following potential causes and solutions:

• Multiple bands or bands at unexpected molecular weights:

  • Alternative splicing: DMC1 has alternative spliced forms, including DMC1-D identified in germ cells

  • Post-translational modifications: Phosphorylation or other modifications may alter migration

  • Degradation products: Add fresh protease inhibitors to lysate preparation

  • Cross-reactivity: Validate with knockout/knockdown controls

• No bands or weak signal:

  • Low expression: DMC1 expression is restricted to meiotic cells; confirm sample type

  • Inefficient transfer: Check transfer efficiency with Ponceau S staining

  • Antibody concentration: Try higher primary antibody concentration (e.g., 1/250 instead of 1/500)

  • Detection sensitivity: Use enhanced chemiluminescence substrates or increase exposure time

• High background:

  • Blocking inefficiency: Increase blocking time or change blocking agent

  • Antibody specificity: Use more stringent washing conditions

  • Membrane issues: Replace membrane if it was allowed to dry during processing

• Expected band size:

  • Human DMC1 should appear at approximately 38 kDa

  • Confirm molecular weight markers are functioning properly

  • Consider running positive control samples with known DMC1 expression

When troubleshooting, methodically change one variable at a time and document all modifications to identify the specific factor affecting your results.

How can I improve the signal-to-noise ratio in DMC1 immunofluorescence?

Improving signal-to-noise ratio in DMC1 immunofluorescence requires optimization of several parameters:

• Fixation optimization:

  • Test different fixatives (paraformaldehyde, methanol, etc.)

  • Optimize fixation time and temperature

  • For meiotic cells, fixation in 40% EtOH with 0.1 M Sorbitol has been effective

• Antibody dilution optimization:

  • Test a range of primary antibody dilutions around the recommended 1/100

  • Optimize secondary antibody concentrations independently

  • Use antibody diluents with background-reducing components

• Blocking strategies:

  • Increase blocking time or concentration

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Include detergents (0.1-0.3% Triton X-100) to reduce non-specific binding

• Washing protocols:

  • Increase number and duration of washes

  • Use PBS with 0.1% Tween-20 (PBST) for more effective washing

  • Ensure complete wash buffer removal between steps

• Counterstaining considerations:

  • Add NP40 (0.5%) before DAPI staining to enhance nuclear penetration

  • Optimize DAPI concentration (typically 0.1 mg/ml) to avoid oversaturation

• Microscopy settings:

  • Optimize exposure settings to prevent saturation

  • Use appropriate filters to minimize autofluorescence

  • Consider confocal microscopy for improved signal-to-noise ratio

For meiotic studies specifically, chromosome spreading techniques often provide superior results compared to whole-cell immunofluorescence due to improved chromatin accessibility and reduced cytoplasmic background.

What strategies can resolve inconsistent results in DMC1 co-immunoprecipitation experiments?

Inconsistent co-immunoprecipitation results with DMC1 antibodies can be addressed through several strategic approaches:

• Buffer optimization:

  • Salt concentration: Maintain physiological (150 mM) NaCl concentrations for native interactions

  • Detergent type and concentration: Test milder detergents for preserving weak interactions

  • pH optimization: Test different pH values to identify optimal interaction conditions

• Timing considerations:

  • For meiotic studies, timing is critical as DMC1-Mlh3 interaction varies across meiotic progression

  • Strong interaction is observed at 6 hours post-induction, with weaker signal at 4 hours

  • Culture volume affects synchronization (larger volumes show ~2 hour delays)

• Control for DNA-mediated interactions:

  • Add DNase I (2 units) with MgCl₂ (2.5 mM) and incubate at 37°C

  • Verify DNA degradation by treating a sample aliquot with Proteinase K and running on agarose gel

  • True protein-protein interactions should persist after DNase treatment

• Antibody selection and orientation:

  • Test different antibodies (monoclonal vs. polyclonal)

  • Reverse the IP approach (pull down with partner protein antibody)

  • Consider epitope tags for clean pull-downs when possible

• Technical considerations:

  • Use protein G immunoprecipitation kits for consistent results

  • For FLAG-tagged proteins, maintain consistent protein amounts (2 μg recombinase with 1 μg MLH complex)

  • Standardize incubation times (30 minutes at 4°C is recommended)

By systematically addressing these factors, researchers can improve the consistency and reliability of DMC1 co-immunoprecipitation experiments.

How should experimental conditions be modified when comparing DMC1 across different species?

When comparing DMC1 across different species, several modifications to experimental protocols are necessary:

• Antibody selection considerations:

  • Verify epitope conservation across species of interest

  • Species-specific antibodies may be required as demonstrated by yeast Mlh1-Mlh3 interacting with yeast DMC1 but not human DMC1

  • For cross-species studies, use antibodies raised against highly conserved regions

• Sequence homology assessment:

  • Perform sequence alignment to identify conserved and variable regions

  • Target conserved epitopes for cross-species antibody selection

  • Predict potential cross-reactivity issues based on homology

• Protocol adjustments for cell/tissue preparation:

  • Species-specific fixation protocols may be required

  • Extraction buffers may need optimization for different tissue types

  • Incubation times and temperatures may require adjustment

• Control validation strategy:

  • Include species-specific positive controls

  • Validate antibody specificity in each species independently

  • Consider using tagged proteins for direct comparison across species

• Interaction studies modifications:

  • For protein-protein interaction studies, consider species compatibility

  • Test interactions within and between species (e.g., human DMC1 with mouse partners)

  • Interpret negative results cautiously as they may reflect species-specific interaction surfaces rather than absence of interaction

These considerations ensure valid cross-species comparisons while accounting for the inherent biological variations in DMC1 structure and function across different organisms.

How are DMC1 antibodies contributing to understanding fertility disorders?

DMC1 antibodies are instrumental in advancing our understanding of fertility disorders through several research approaches:

• Analysis of meiotic defects in infertility:

  • DMC1 antibodies enable visualization of recombination intermediates in testicular biopsies

  • Abnormal DMC1 staining patterns may indicate recombination defects leading to infertility

  • Quantitative analysis of DMC1 foci can reveal subtle meiotic abnormalities

• Genetic variant functional studies:

  • DMC1 antibodies help assess the impact of DMC1 variants identified in infertile patients

  • Immunofluorescence can reveal altered localization or focus formation

  • Co-immunoprecipitation can identify disrupted protein interactions in variant proteins

• Model organism research:

  • DMC1 antibodies facilitate studies in mice and other model organisms

  • Comparative analysis between human samples and model systems

  • Antibodies detecting mouse, rat, and human DMC1 enable translational research

• Therapeutic target investigation:

  • DMC1 antibodies help evaluate potential interventions targeting meiotic recombination

  • Assessment of drug effects on DMC1 localization and function

  • Monitoring DMC1 dynamics in response to hormonal treatments

These applications of DMC1 antibodies contribute to diagnosing the molecular basis of unexplained infertility and developing potential therapeutic strategies for reproductive medicine.

What novel insights have DMC1 antibodies provided about cancer-related genomic instability?

While DMC1 is primarily associated with meiotic recombination, DMC1 antibodies have revealed unexpected connections to cancer biology:

• Aberrant expression in cancer cells:

  • DMC1 antibodies have detected expression in cancer cell lines including Raji (Burkitt's lymphoma) and K562 (chronic myelogenous leukemia)

  • Immunocytochemistry has shown DMC1 presence in HeLa cells (cervical adenocarcinoma)

  • This unexpected expression suggests potential roles in genomic instability in cancer

• Relationship to DNA repair mechanisms:

  • DMC1's homology to RAD51 suggests potential roles in alternative repair pathways

  • DMC1 antibodies help distinguish between RAD51-dependent and potential DMC1-involved repair

  • Understanding these mechanisms may explain therapy resistance in some cancers

• Associations with chromosomal rearrangements:

  • DMC1 antibodies can identify potential involvement in cancer-associated chromosomal translocations

  • Aberrant recombination activity may contribute to oncogenic structural variants

  • Co-localization studies with cancer-specific breakpoint regions provide mechanistic insights

• Potential therapeutic implications:

  • DMC1 detection in cancer cells suggests potential as a biomarker or therapeutic target

  • Antibody-based screening could identify cancers with aberrant DMC1 expression

  • Correlation of expression with treatment outcomes may guide personalized therapy approaches

These findings challenge the traditional view of DMC1 as exclusively meiotic and suggest broader roles in genomic maintenance that may be relevant to cancer development and treatment.

How can DMC1 antibodies be integrated with emerging genomic and imaging technologies?

Integration of DMC1 antibodies with cutting-edge technologies is expanding research possibilities:

• Combination with super-resolution microscopy:

  • STORM/PALM microscopy with DMC1 antibodies offers nanoscale visualization of recombination nodules

  • Enhanced resolution can reveal previously undetectable structural features

  • Multi-color approaches allow simultaneous visualization of multiple recombination proteins

• Integration with ChIP-seq and CUT&RUN:

  • DMC1 antibodies enable chromatin immunoprecipitation followed by sequencing

  • Genome-wide mapping of DMC1 binding sites identifies recombination hotspots

  • CUT&RUN with DMC1 antibodies provides higher resolution with less background

• Single-cell approaches:

  • DMC1 antibodies in single-cell imaging allow heterogeneity analysis in meiotic progression

  • Correlation with single-cell transcriptomics or proteomics

  • Reveals cell-to-cell variability in recombination processes

• Live-cell imaging applications:

  • DMC1 antibody fragments (Fabs) or nanobodies for live-cell dynamics

  • Real-time visualization of recombination nodule assembly and disassembly

  • Correlation with chromosome movement during meiotic prophase

• Proximity labeling techniques:

  • DMC1 antibodies in combination with BioID or APEX2 proximity labeling

  • Identification of novel DMC1-proximal proteins during meiotic recombination

  • Temporal mapping of the changing DMC1 interactome throughout meiosis

These integrative approaches provide multi-dimensional insights into DMC1 function that extend beyond traditional antibody applications.

What are the implications of recent DMC1-MLH3 interaction studies for understanding meiotic crossover regulation?

Recent discoveries of DMC1-MLH3 interactions have significant implications for our understanding of meiotic crossover regulation:

• Physical interaction evidence:

  • Co-immunoprecipitation experiments have confirmed that Mlh3 physically interacts with Dmc1 during meiotic prophase

  • This interaction is detected most strongly at 6 hours post-meiotic induction, with weaker signal at 4 hours

  • The interaction persists after Benzonase treatment, confirming it is not mediated through DNA

• Molecular mechanistic insights:

  • The interaction suggests direct communication between early recombination (DMC1) and crossover resolution (MLH3)

  • This challenges the traditional sequential model of recombination where these proteins act in temporally distinct phases

  • Implies potential feedback mechanisms between early and late recombination events

• Evolutionary conservation analysis:

  • Yeast Mlh1-Mlh3 robustly immunoprecipitates yeast Dmc1 and yeast Rad51 but not human DMC1

  • This species-specificity suggests evolved differences in recombination regulation

  • Yeast Mlh1-Pms1 also immunoprecipitates yeast Dmc1, indicating Mlh1 may mediate these interactions

• Crossover assurance implications:

  • Early DMC1-MLH3 interaction may help establish crossover designation

  • Could explain mechanisms ensuring at least one crossover per chromosome pair

  • May provide insights into crossover interference (spacing) regulation

These findings represent a significant advance in our understanding of the molecular mechanisms governing meiotic recombination and highlight the complexity of protein interactions ensuring accurate genetic exchange during gamete formation.