MRE11A Antibody

What is MRE11A and why is it important in cellular research?

MRE11A (Meiotic Recombination 11 Homolog A) is a core component of the MRN complex (MRE11/RAD50/NBS1), which plays a central role in double-strand break (DSB) repair, DNA recombination, maintenance of telomere integrity, and meiosis. MRE11A provides the complex with both single-strand endonuclease activity and double-strand-specific 3'-5' exonuclease activity . This protein is critical in DNA damage response pathways, as it initiates end resection at DSB sites by cooperating with CtIP to prevent non-homologous end joining (NHEJ) and license homologous recombination (HR) . Deficient activity of MRE11A has been linked to premature T cell aging, tissue-invasiveness in rheumatoid arthritis, and various DNA repair disorders .

What applications are MRE11A antibodies validated for?

MRE11A antibodies have been validated for multiple experimental applications:

The optimal dilution should be determined by each laboratory for specific applications and sample types .

How can I validate the specificity of an MRE11A antibody in my experimental system?

To validate MRE11A antibody specificity:

• Positive controls: Use cell lines with confirmed MRE11A expression (HeLa, HEK-293, Jurkat, K-562)

• Knockdown validation: Employ siRNA targeting MRE11A and confirm reduction in signal intensity

• Overexpression validation: Use MRE11A-overexpressing constructs to confirm increased signal intensity

• Immunoprecipitation-Western blot: Perform IP followed by WB with a different MRE11A antibody

• Cross-reactivity assessment: Test antibody against recombinant MRE11A and related proteins

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm signal specificity

These methods provide complementary evidence for antibody specificity and should be documented in research publications.

What are the optimal sample preparation methods for detecting MRE11A in different cellular compartments?

MRE11A localization varies based on cellular state and stimuli. For comprehensive detection:

Nuclear Fraction:

• Use nuclear extraction buffer containing 20mM HEPES (pH 7.9), 1.5mM MgCl₂, 0.42M NaCl, 0.2mM EDTA, and 25% glycerol

• Add protease inhibitors, phosphatase inhibitors, and 1mM DTT fresh before use

• For chromatin-bound MRE11A, perform additional DNase treatment or salt extraction

Chromatin Fraction:

• After nuclear isolation, treat with nuclease to release chromatin-bound proteins

• Alternative approach: sequential salt extraction (100mM, 300mM, 420mM NaCl)

• Note that MRE11A recruitment to chromatin increases significantly after DNA damage induction

Telomere-Associated Detection:

• For telomere-bound MRE11A, dual-color immunostaining with anti-MRE11A and anti-TRF2 antibodies is recommended

• This technique revealed that RA T cells have telomeric tips almost depleted of MRE11A

What controls should be included when using MRE11A antibodies in DNA damage response studies?

When studying DNA damage responses with MRE11A antibodies:

• Positive damage control: Include samples treated with known DNA-damaging agents (e.g., MNNG at 50-150nM, ionizing radiation, or etoposide)

• Temporal controls: Collect samples at multiple time points post-damage induction (immediate, 12h, 24h, 48h)

• Subcellular localization control: Compare cytoplasmic, nuclear, and chromatin fractions

• Co-localization controls: Include antibodies against other MRN complex components (RAD50, NBS1) and DNA damage markers (γH2AX, 53BP1)

• Functional controls: Include inhibitors of ATM kinase to distinguish MRE11A function from downstream signaling

• Cell cycle controls: Synchronize cells or use cell cycle markers to account for cell cycle-dependent changes in MRE11A function

Research has shown that MRE11A is recruited to chromatin within 12 hours of MNNG treatment, demonstrating its involvement in processing alkylation damage .

How can MRE11A antibodies be used to study the relationship between telomere damage and T cell aging?

MRE11A deficiency has been linked to premature T cell aging through telomere deprotection. To investigate this:

• Telomeric damage assessment: Perform dual immunostaining with anti-MRE11A and anti-TRF2 antibodies to quantify MRE11A localization at telomeres

• Telomeric damage foci: Use co-staining of MRE11A with 53BP1 and TRF2 to identify telomeric damage foci

• Quantitative analysis: Measure colocalization coefficients between MRE11A and TRF2 signals

• Comparative analysis: Compare T cells from healthy donors versus patients with autoimmune conditions like rheumatoid arthritis

• Functional rescue experiments: Perform MRE11A overexpression in deficient cells to demonstrate reversal of telomeric damage

• Downstream marker analysis: Measure senescence markers (p16, p21, CD57) in relation to MRE11A expression

Research has shown that MRE11A-low T cells display telomeric damage foci formation and upregulation of senescence markers. Restoring MRE11A expression reduced 53BP1 and TRF2 colocalization by 75%, demonstrating direct involvement in telomere repair .

What methodological approaches can resolve contradictory data when studying MRE11A in different experimental systems?

When facing contradictory MRE11A data across different systems:

• Antibody validation matrix:

  • Test multiple antibodies targeting different epitopes of MRE11A

  • Compare monoclonal vs. polyclonal antibodies

  • Validate each antibody in both control and experimental systems

• Species-specific considerations:

  • Human MRE11A and mouse Mre11a share 84% homology

  • Rat Mre11a shows 94% homology to mouse

  • Use species-specific positive controls and validate cross-reactivity

• Cell type variations:

  • Different cell types show varying MRE11A expression levels

  • Compare expression in primary cells vs. cell lines

  • Normalize to appropriate housekeeping proteins

• Functional validation:

  • Complement protein detection with nuclease activity assays

  • Use MRE11A inhibitors (e.g., Mirin) as functional controls

  • Perform genetic knockdown/knockout with multiple approaches (siRNA, shRNA, CRISPR)

• Context-dependent regulation:

  • MRE11A can have opposing functions in different DNA repair pathways

  • For example, MRE11A acts as a negative regulator of DNA mismatch repair while being essential for double-strand break repair

How can MRE11A antibodies be optimized for chromatin immunoprecipitation (ChIP) studies of DNA damage sites?

ChIP optimization for MRE11A requires specific considerations:

• Crosslinking optimization:

  • Test both formaldehyde (1% for 10 min) and dual crosslinkers (DSG followed by formaldehyde)

  • Optimize crosslinking time based on protein-DNA interaction strength

• Chromatin preparation:

  • Sonication parameters: 10-15 cycles (30 sec ON/30 sec OFF) to achieve 200-500 bp fragments

  • Alternative: enzymatic digestion with micrococcal nuclease

  • Verify fragment size by agarose gel electrophoresis

• Antibody selection and validation:

  • Pre-clear chromatin with protein A/G beads

  • Use antibodies validated specifically for ChIP applications

  • Include IgG control and positive control antibody (e.g., histone H3)

• DNA damage induction for peak enrichment:

  • Treat cells with DNA damaging agents (ionizing radiation, etoposide, or MNNG)

  • Perform time course (30 min to 4 h post-damage) to capture optimal MRE11A recruitment

  • Use γH2AX ChIP as positive control for damage sites

• Sequential ChIP (re-ChIP):

  • For studying MRN complex assembly, perform sequential ChIP with antibodies against MRE11A followed by RAD50 or NBS1

  • Include appropriate controls for each immunoprecipitation step

Research has shown that MLH1 deficiency, but not MSH2 deficiency, significantly decreases chromatin-bound MRE11A, highlighting the importance of considering interacting partners .

How can MRE11A antibodies be used to investigate its role in rheumatoid arthritis pathogenesis?

Research has identified MRE11A deficiency as a contributor to pathogenic T cell behavior in rheumatoid arthritis (RA):

• Comparative expression analysis:

  • Compare MRE11A levels in T cells from RA patients versus healthy controls and disease controls (e.g., PsA)

  • Use flow cytometry with intracellular staining to quantify MRE11A in different immune cell subsets

  • Analyze both naive and memory CD4+ T cell populations separately

• Subcellular localization studies:

  • Perform dual-color immunostaining with anti-MRE11A and anti-TRF2 to assess telomeric MRE11A

  • Quantify nuclear and telomere-bound MRE11A in patient-derived cells

• Functional rescue experiments:

  • Overexpress MRE11A in RA T cells using appropriate constructs

  • Assess reversal of pathogenic phenotypes (telomeric damage, senescence markers, invasiveness)

  • Measure inflammatory cytokine production before and after MRE11A restoration

• In vivo validation:

  • Use human-synovium chimeric mouse models

  • Transfer control or MRE11A-overexpressing T cells

  • Analyze tissue invasion and inflammatory marker expression

Studies have shown that MRE11A-low T cells from RA patients are tissue-invasive and pro-arthritogenic in mouse models, while MRE11A reconstitution mitigated synovitis and reduced expression of inflammatory cytokines (TNF, IL6, IL1B) .

What are the methodological approaches for using MRE11A antibodies in cancer research?

MRE11A plays complex roles in cancer biology, requiring specialized methodological approaches:

• Cancer-specific expression profiling:

  • Compare MRE11A levels across tumor types and matched normal tissues

  • Use tissue microarray (TMA) with standardized IHC protocols

  • Score intensity (0-3+) and percentage of positive cells

• Mutation and functional impact assessment:

  • Combine antibody detection with sequencing data

  • Use antibodies recognizing specific domains to detect truncated forms

  • Assess nuclease activity in correlation with protein levels

• Therapy response biomarker studies:

  • Monitor MRE11A levels before and after DNA-damaging therapies

  • Correlate with treatment response and resistance mechanisms

  • Analyze in paired samples (pre- and post-treatment biopsies)

• Double-strand break repair capacity:

  • Use DNA damage induction followed by time-course analysis of repair

  • Track MRE11A recruitment to damage sites using immunofluorescence

  • Correlate with genomic instability markers

• Combination with DNA damage markers:

  • Multiplex immunostaining for MRE11A with γH2AX, 53BP1, RAD51

  • Analyze spatial relationships between markers in tumor sections

  • Correlate patterns with clinical outcomes

MRE11A deficiency can impact cancer treatment response by affecting DNA repair pathways, making it an important biomarker for personalized cancer therapy approaches.

How can MRE11A antibodies help investigate its role in DNA mismatch repair (MMR) regulation?

Recent research has identified MRE11A as a negative regulator of DNA mismatch repair, requiring specific methodological approaches:

• MMR protein interaction studies:

  • Perform co-immunoprecipitation with MRE11A antibodies followed by Western blot for MMR proteins (MLH1, PMS2, MSH2, MSH6)

  • Use proximity ligation assay (PLA) to detect protein-protein interactions in situ

  • Map interaction domains using truncated protein constructs

• Chromatin recruitment analysis:

  • Isolate chromatin fractions after DNA damage induction (MNNG treatment)

  • Compare MRE11A recruitment kinetics with MMR proteins

  • Use MLH1-deficient and MSH2-deficient cells as controls

• MMR activity assays:

  • Implement GFP-heteroduplex repair assays with MRE11A knockdown or overexpression

  • Normalize to transfection efficiency using co-transfected mCherry

  • Quantify repair efficiency under different conditions

• Competition binding studies:

  • Test if MRE11A competes with PMS2 for binding to MLH1

  • Measure PMS2 levels in whole cells and on chromatin with varying MRE11A levels

  • Perform in vitro binding assays with purified proteins

Research has demonstrated that knockdown of MRE11A increases MMR activity, while overexpression decreases it. Mechanistically, MRE11A competes with PMS2 for binding to MLH1, decreasing PMS2 levels and consequently compromising MMR activity .

What strategies can resolve weak or inconsistent MRE11A detection in Western blot?

When facing detection challenges:

• Sample preparation optimization:

  • Use fresh samples or properly stored frozen samples

  • Include protease inhibitors (PMSF, aprotinin, leupeptin, pepstatin A)

  • Add phosphatase inhibitors to preserve phosphorylated forms

  • Test different lysis buffers (RIPA vs. NP-40 vs. specialized nuclear extraction)

• Protein loading and transfer optimization:

  • Increase protein loading (50-100 μg total protein)

  • Optimize transfer conditions (wet transfer at 30V overnight at 4°C for large proteins)

  • Use PVDF membrane instead of nitrocellulose for better protein retention

  • Verify transfer efficiency with reversible staining (Ponceau S)

• Antibody optimization:

  • Test different antibody dilutions (start with 1:1000 and adjust)

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

  • Try different antibodies targeting different epitopes

  • Use signal enhancement systems (biotin-streptavidin amplification)

• Signal detection enhancement:

  • Use high-sensitivity chemiluminescent substrates

  • Extend exposure time in increments

  • Try fluorescent secondary antibodies for increased sensitivity and linearity

• Positive control inclusion:

  • Use cell lines with confirmed high MRE11A expression (HeLa, HEK-293)

  • Include MRE11A-overexpressing lysate as strong positive control

How can I optimize dual immunofluorescence staining of MRE11A with other DNA repair proteins?

For successful co-localization studies:

• Fixation optimization:

  • Test different fixatives: 4% paraformaldehyde (10 min), methanol (-20°C, 10 min), or combination fixation

  • For nuclear proteins, pre-extraction with 0.5% Triton X-100 before fixation can reduce cytoplasmic background

• Antigen retrieval methods:

  • Heat-mediated: citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0)

  • Enzymatic: proteinase K (minimal digestion time)

  • Test which method works best for preserving both antigens

• Antibody compatibility assessment:

  • Verify primary antibodies are from different host species

  • If same species is unavoidable, use directly conjugated antibodies or sequential staining with Fab fragments

  • Test each antibody individually before combining

• Cross-reactivity minimization:

  • Increase blocking time (2h at room temperature with 5% normal serum)

  • Include 0.1-0.3% Triton X-100 in blocking buffer for nuclear proteins

  • Use highly cross-adsorbed secondary antibodies

• Signal amplification for low-abundance targets:

  • Tyramide signal amplification (TSA) for weak signals

  • Avoid over-amplification which can obscure co-localization

• Advanced imaging and analysis:

  • Use high-resolution confocal microscopy with appropriate controls

  • Employ deconvolution algorithms to improve signal-to-noise ratio

  • Calculate co-localization coefficients (Pearson's, Manders') for quantitative analysis

Research has successfully used dual-color immunostaining with anti-MRE11A and anti-TRF2 to quantify telomere-bound MRE11A in T cells .

What are the key considerations for generating and validating MRE11A knockdown controls?

To create reliable knockdown controls:

• siRNA design and selection:

  • Design 2-3 different siRNA sequences targeting different regions of MRE11A mRNA

  • Use algorithms to predict efficiency and minimize off-target effects

  • Include non-targeting control siRNA with similar GC content

• Transfection optimization:

  • Determine optimal cell density (typically 50-70% confluence)

  • Test multiple transfection reagents and conditions

  • Establish transfection efficiency using fluorescent control siRNA

• Knockdown validation methods:

  • Western blot: Quantify protein reduction (typically 48-72h post-transfection)

  • qRT-PCR: Measure mRNA reduction (typically 24-48h post-transfection)

  • Immunofluorescence: Visualize protein reduction at single-cell level

• Functional validation:

  • Confirm expected phenotypes (e.g., increased sensitivity to DNA-damaging agents)

  • Measure DNA damage marker changes (increased 53BP1 foci, CHK1 phosphorylation)

  • Rescue experiments by expressing siRNA-resistant MRE11A constructs

• Off-target effect assessment:

  • Compare phenotypes across multiple siRNA sequences

  • Perform transcriptome analysis to identify potential off-target effects

  • Validate key findings with genetic knockout approaches