eepd1 Antibody

What is EEPD1 and why is it important in molecular biology research?

EEPD1 is a 569 amino acid protein characterized by two amino-terminal helix-hairpin-helix (HhH) DNA binding domains related to RuvA, and a carboxy-terminal DNase I-like domain that places it in the exonuclease-endonuclease-phosphatase (EEP) family . EEPD1 plays crucial roles in:

• Repair of oxidatively-stressed replication forks

• Maintenance of genome stability during replication stress

• Promotion of homologous recombination (HR) repair while inhibiting classical non-homologous end joining (cNHEJ)

• Enhancement of 5' DNA end resection

• Facilitation of stressed replication fork restart

Understanding EEPD1 function is important for research in genome stability, DNA damage response, and cancer biology, as EEPD1 is overexpressed in certain cancers including colorectal cancers and large cell lymphomas .

What applications are EEPD1 antibodies suitable for?

Based on commercial availability and published research, EEPD1 antibodies have been validated for several applications:

When selecting an antibody, researchers should consider the specific application needs and available validation data for their experimental system .

What is the subcellular localization of EEPD1 and how does this affect antibody selection?

The subcellular localization of EEPD1 is complex and context-dependent:

• EEPD1 has been reported to localize to the plasma membrane, dependent on N-terminal lipid modifications (myristoylation and palmitoylation)

• It can be recruited to stalled replication forks following DNA damage

• Nuclear localization has been observed in some studies, particularly in DNA damage contexts

• It may localize differently depending on cell type and experimental conditions

When selecting an antibody for studying EEPD1 localization, researchers should:

• Choose antibodies validated for immunofluorescence or immunocytochemistry

• Consider using subcellular fractionation approaches followed by Western blotting to confirm localization patterns

• Be aware that fixation and permeabilization methods may affect epitope accessibility, especially for membrane-bound forms

What are the optimal protocols for detecting EEPD1 by Western blotting?

For optimal Western blot detection of EEPD1:

• Sample preparation:

  • Include protease inhibitors in lysis buffers

  • For chromatin-bound EEPD1, isolate chromatin fractions using established protocols

  • For membrane-associated EEPD1, prepare crude membrane fractions

• Gel electrophoresis and transfer:

  • Use 8-10% SDS-PAGE gels as EEPD1 has a predicted molecular weight of 62-63 kDa

  • Allow sufficient separation time as EEPD1 may run close to other proteins of similar size

• Antibody incubation:

  • Primary antibody dilutions: 1:2000-1:10000 (optimize for specific antibody)

  • Include appropriate controls (see section 2.3)

  • Expected band size: 62-63 kDa

• Detection considerations:

  • EEPD1 may show variable expression levels across cell types

  • DNA damage treatments may increase EEPD1 recruitment to chromatin fractions

How can I design experiments to study EEPD1's role in DNA repair?

To investigate EEPD1's functions in DNA repair mechanisms:

• Gene expression modulation approaches:

  • siRNA transfection: Use Lipofectamine RNAi-Max with 30 nM siRNA, with incubation for 24-48 hours

  • CRISPR-Cas9 for genetic deletion: HeLa cell lines with EEPD1 deletion have been reported

  • Rescue experiments: Express siRNA-resistant EEPD1 to confirm specificity of phenotypes

• DNA damage induction protocols:

  • Oxidative stress: 1 mM H₂O₂ treatment for 1 hour

  • Replication stress: 10-18 hour exposure to 10 μM VP-16 or hydroxyurea treatment

  • Other agents: camptothecin, UV light, cisplatin, ionizing radiation

• Functional assays:

  • Clonogenic survival assays to assess cell viability after DNA damage

  • DNA end resection measurement using BrdU incorporation in non-denatured ssDNA

  • Replication fork restart assays using BrdU or EdU pulse-labeling

  • Chromosomal abnormality quantification

  • DSB repair pathway choice using reporter systems (HR vs. NHEJ)

• Protein interaction studies:

  • Co-immunoprecipitation to detect interactions with Exo1, CtIP, BLM, RPA32

  • Chromatin immunoprecipitation to assess recruitment to damage sites

What controls should be included when using EEPD1 antibodies?

Proper experimental controls are essential for interpreting results with EEPD1 antibodies:

• Negative controls:

  • EEPD1 knockout or knockdown samples (siRNA or CRISPR)

  • IgG isotype controls for immunoprecipitation and ChIP experiments

  • Secondary antibody-only controls for immunofluorescence

• Positive controls:

  • Cells with known EEPD1 expression (COLO 320 cells, PC-3 cells have been used)

  • EEPD1 overexpression samples

  • DNA damage treatment to increase chromatin-bound EEPD1

• Specificity controls:

  • Peptide competition assays

  • Multiple antibodies targeting different epitopes

  • Signal verification in multiple cell lines

• Loading controls:

  • For whole cell lysates: standard housekeeping proteins

  • For chromatin fractions: Histone H3

  • For membrane fractions: appropriate membrane protein markers

How can EEPD1 antibodies be used to study protein interactions in DNA repair pathways?

EEPD1 functions in DNA repair through interactions with multiple proteins. To study these interactions:

• Co-immunoprecipitation (Co-IP) approaches:

  • EEPD1 has been shown to interact with Exo1, CtIP, BLM, and RPA32

  • Perform reciprocal Co-IPs validating interactions in both directions

  • Consider crosslinking approaches for transient interactions

  • Include nuclease treatments to determine if interactions are DNA-dependent

• Proximity ligation assay (PLA):

  • For visualizing protein-protein interactions in situ

  • Particularly useful for studying interactions at replication forks or DNA damage sites

• Chromatin IP (ChIP) and iPOND:

  • EEPD1 is recruited to stalled replication forks and DNA break sites

  • iPOND has been used to study EEPD1 recruitment to nascent DNA at replication forks

  • ChIP can assess EEPD1 recruitment to specific genomic loci after DSB induction

• Protein complex stability analysis:

  • Interestingly, depletion of EEPD1 leads to degradation of Exo1 and BLM, suggesting they form an obligate complex

  • Western blotting can assess stability of interaction partners after EEPD1 depletion

What methodologies can reveal EEPD1's enzymatic activities?

EEPD1 exhibits nuclease activities relevant to its role in DNA repair:

• Nuclease activity assays:

  • EEPD1 shows 5' overhang nuclease activity

  • It also possesses 5' abasic endonuclease activity similar to APE1

  • In vitro assays can be performed using 32P-labeled oligonucleotides with varying structures (fork structures, abasic sites)

• Experimental setup for activity assays:

  • Reactions typically include: buffer containing 50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 0.5 mM DTT, 50 mM NaCl, 0.1 mg/ml BSA, 2% glycerol, and 1 μg poly(dI-dC)

  • Recombinant EEPD1 protein (100-300 ng, 2.5-7.5 pmol)

  • 32P-labeled DNA substrates

  • Incubation for 20 minutes at room temperature

  • Analysis by non-denaturing polyacrylamide gel electrophoresis

• Substrate specificity determination:

  • Competition experiments using unlabeled identical oligonucleotides (10×, 25×, and 50× molar excess)

  • Various DNA structures including control oligomers, fork-DNA structures, and abasic site-containing substrates

How can I investigate EEPD1's involvement in repair pathway choice during replication stress?

EEPD1 plays a critical role in directing repair pathway choice at stalled replication forks:

• Repair pathway reporter assays:

  • HR reporter systems (e.g., HT256) show a 6.4-fold reduction in HR upon EEPD1 depletion

  • cNHEJ reporter systems (e.g., EJ5) show a 2.3-fold increase in cNHEJ upon EEPD1 depletion

  • MMEJ/HR-MluI reporter system reveals that EEPD1 promotes HR while inhibiting MMEJ

• Gene conversion tract analysis:

  • EEPD1 depletion results in longer gene conversion tracts in HR products

  • This suggests EEPD1 is important for stable heteroduplex formation during HR

• Genetic interaction studies:

  • 53BP1 depletion rescues cytogenetic abnormalities caused by EEPD1 depletion

  • Co-depletion experiments with Exo1 or CtIP suggest EEPD1 functions in the same resection pathway

• Microscopy approaches:

  • Immunofluorescence analysis of repair factor foci formation (RAD51, γ-H2AX, phospho-RPA32) after replication stress is reduced with EEPD1 depletion

  • Time-course experiments can track the kinetics of repair pathway activation

Why might I observe variability in EEPD1 detection across experiments?

Variability in EEPD1 detection can result from several factors:

• Subcellular localization issues:

  • EEPD1 localizes to different cellular compartments (membrane, nucleus, chromatin) depending on context

  • Extraction methods may not efficiently capture all pools of EEPD1

  • Solution: Use subcellular fractionation approaches to isolate specific compartments

• Expression level variations:

  • EEPD1 expression varies across tissues and cell lines

  • Expression may be induced by DNA damage or replication stress

  • Solution: Normalize to appropriate loading controls and include positive control cell lines

• Post-translational modifications:

  • N-terminal lipid modifications (myristoylation and palmitoylation) affect localization

  • Potential phosphorylation during DNA damage response

  • Solution: Consider using phosphatase inhibitors in lysis buffers and antibodies that recognize specific modified forms

• Protein complex formation:

  • EEPD1 forms complexes with other proteins (Exo1, BLM)

  • These interactions may mask epitopes

  • Solution: Try different antibodies targeting different epitopes

How can I optimize immunofluorescence protocols for EEPD1 detection?

For successful immunofluorescence detection of EEPD1:

• Fixation optimization:

  • PFA fixation (1-4%) works for detection in U2 OS cells

  • For membrane-associated EEPD1, avoid methanol fixation which can disrupt membrane structures

  • Consider mild fixation conditions to preserve epitope accessibility

• Permeabilization considerations:

  • Triton X-100 permeabilization has been used successfully

  • For membrane-bound forms, gentler detergents like saponin may better preserve localization

• Antibody selection and dilution:

  • Use antibodies validated for IF applications (e.g., 4 μg/ml concentration has been reported)

  • Include appropriate blocking steps to reduce background

  • Consider using fluorescently-conjugated EEPD1 antibodies for direct detection

• Co-staining strategies:

  • Co-stain with markers of different cellular compartments

  • For DNA damage studies, co-stain with γ-H2AX, RAD51, or 53BP1

  • For replication studies, consider EdU labeling of nascent DNA

How should I interpret changes in EEPD1 localization after DNA damage?

EEPD1 dynamics during DNA damage response:

• Recruitment patterns:

  • EEPD1 is recruited to stalled replication forks after hydroxyurea or H₂O₂ treatment

  • iPOND analysis shows association with nascent DNA at stressed forks

  • ChIP experiments demonstrate recruitment to specific DSB sites

• Temporal considerations:

  • Peak chromatin association occurs within 0-2 hours after release from replication stress

  • This coincides with the time of maximal replication fork restart

• Relationship to other repair factors:

  • EEPD1 is required for proper formation of RAD51, γ-H2AX, and phospho-RPA32 foci

  • It acts upstream of ATR and CHK1 phosphorylation

  • NBS1, 53BP1, and BRCA1 foci formation are not dependent on EEPD1

• Functional significance:

  • Altered EEPD1 localization reflects its active involvement in repair processes

  • Failure to relocalize may indicate defects in DNA damage signaling

  • Cytogenetic abnormalities resulting from EEPD1 deficiency include nuclear bridges and micronuclei

What are promising approaches for studying EEPD1 in cancer biology?

EEPD1's role in DNA repair makes it interesting in cancer research contexts:

• Expression analysis opportunities:

  • EEPD1 is overexpressed in colorectal cancers and large cell lymphomas

  • Researchers can analyze expression patterns across cancer types using:

    • Immunohistochemistry with specific EEPD1 antibodies

    • Correlation of expression with patient outcomes and treatment response

• Therapeutic targeting potential:

  • EEPD1 inhibition could sensitize tumors to replication stress-inducing chemotherapeutics

  • Synthetic lethality approaches with other DNA repair defects

  • Investigation of cell death mechanisms when EEPD1 is inhibited in cancer cells

• Biomarker development:

  • EEPD1 expression or phosphorylation status as potential predictive biomarkers

  • Correlation with response to DNA-damaging agents in different cancer types

• Experimental models:

  • Cell line panels with varying EEPD1 expression levels

  • Patient-derived xenografts

  • In vivo studies examining effects of EEPD1 modulation on tumor growth and treatment response

How can advanced imaging techniques enhance our understanding of EEPD1 dynamics?

New imaging approaches can reveal EEPD1 behavior at unprecedented resolution:

• Live-cell imaging approaches:

  • Fluorescently-tagged EEPD1 to track recruitment to damage sites in real-time

  • FRAP (Fluorescence Recovery After Photobleaching) to measure protein mobility

  • Photoactivatable or photoconvertible EEPD1 fusions to track specific protein populations

• Super-resolution microscopy:

  • STORM/PALM to visualize EEPD1 localization at nanometer resolution

  • Structured illumination microscopy (SIM) to improve visualization of EEPD1 at replication forks

  • Expansion microscopy to physically enlarge specimens for improved resolution

• Correlative light and electron microscopy (CLEM):

  • Combining fluorescence imaging of EEPD1 with ultrastructural context

  • Immunogold labeling for electron microscopy to precisely localize EEPD1

• Multi-protein tracking:

  • Simultaneous imaging of EEPD1 with interaction partners (Exo1, BLM)

  • FRET approaches to measure direct protein-protein interactions