ERCC6 Antibody

What is ERCC6 and why is it significant in molecular biology research?

ERCC6, also known as CSB (Cockayne Syndrome B protein), belongs to the SNF2/RAD54 helicase family and plays a critical role in DNA repair mechanisms. It functions primarily in the preferential repair of active genes through its presumed DNA or RNA unwinding capabilities . The protein is approximately 168 kDa in size (calculated weight), though it typically appears around 150-170 kDa in Western blots due to post-translational modifications .

ERCC6 corrects UV survival and RNA synthesis after UV exposure in Cockayne syndrome complementation group B cells . Additionally, recent research has revealed its importance in chromatin remodeling during DNA repair processes . Its significance extends to multiple human diseases, as defects in ERCC6 are associated with Cockayne syndrome type B, cerebro-oculo-facio-skeletal syndrome type 1, De Sanctis-Cacchione syndrome, and UV-sensitive syndrome . Genetic variations in ERCC6 have also been linked to age-related macular degeneration type 5 .

What types of ERCC6 antibodies are available for research applications?

Research-grade ERCC6 antibodies come in several configurations:

Most commercially available ERCC6 antibodies are polyclonal, with rabbit being the predominant host species . These antibodies target different regions of the ERCC6 protein, including N-terminal domains (AA 5-34), internal regions (AA 201-300, AA 717-731), and C-terminal domains (AA 1394-1493) .

Applications validated for these antibodies include Western blotting (1:200-1:6000 dilution range), immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate), immunohistochemistry on paraffin-embedded and frozen sections, immunocytochemistry, immunofluorescence, and ELISA .

What are the optimal conditions for Western blotting with ERCC6 antibodies?

Successful Western blotting with ERCC6 antibodies requires careful optimization:

For optimal results with Proteintech's 20548-1-AP antibody, use a dilution range of 1:200-1:1000 . For their 24291-1-AP antibody, a dilution range of 1:1000-1:6000 is recommended . The calculated molecular weight of ERCC6 is 168 kDa, but observed molecular weights in SDS-PAGE typically range from 150-170 kDa .

Sample preparation should include complete cell lysis and protein denaturation. Storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For specialized protocols, manufacturers often provide downloadable Western blot protocols specific to their antibodies .

How should I validate ERCC6 antibody specificity for my experimental system?

Rigorous validation of ERCC6 antibodies is essential for reliable experimental results:

• Positive and negative controls:

  • Positive controls: Use cell lines with known ERCC6 expression (HeLa, PC-3, DU 145 cells)

  • Negative controls: Include ERCC6 knockout/knockdown samples

  • Published studies have used siRNA knockdown to validate antibody specificity

• Multi-method validation:

  • Confirm results using at least two different detection techniques (e.g., WB and IF)

  • Recent publications have validated ERCC6 antibodies using a combination of Western blotting, immunoprecipitation, and immunofluorescence

• Cross-reactivity testing:

  • Verify species reactivity if working with non-human models

  • Many ERCC6 antibodies show reactivity with human, mouse, and rat samples

  • Test cross-reactivity empirically even when manufacturer claims compatibility

• Epitope verification:

  • For critical experiments, consider using antibodies targeting different epitopes of ERCC6

  • Various antibodies target different regions from N-terminal to C-terminal domains

Recent studies examining Cockayne syndrome patients have employed Western blotting with ERCC6 antibodies to demonstrate the deleterious effects of novel ERCC6 mutations, providing a good reference for validation approaches .

What are common technical challenges when working with ERCC6 antibodies and how can they be addressed?

Researchers commonly encounter several challenges when working with ERCC6 antibodies:

For weak signals, consider that ERCC6 expression may be cell-type dependent or affected by experimental conditions. PC-3, HeLa, and DU 145 cells have been confirmed to express detectable levels of ERCC6 . For mouse models, brain tissue has shown positive Western blot results .

If experiencing high background, increasing blocking time (using 5% BSA or milk) and optimizing primary antibody dilution can help. Most ERCC6 antibodies are recommended to be stored at -20°C with glycerol and BSA to maintain reactivity .

For multiple bands, verify whether these represent known isoforms or are artifacts. The calculated molecular weight of ERCC6 is 168 kDa, with observed weights typically between 150-170 kDa . Significant deviations from this range may indicate degradation or non-specific binding.

How can ERCC6 antibodies be optimized for immunoprecipitation experiments?

Successful immunoprecipitation of ERCC6 requires careful optimization:

• Antibody selection:

  • Choose antibodies validated for IP applications (e.g., Proteintech's 24291-1-AP)

  • Recommended usage: 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Cell line selection:

  • HeLa cells have been successfully used for ERCC6 immunoprecipitation

  • Choose cell lines with reasonable ERCC6 expression levels

• Lysis conditions:

  • Use gentle lysis buffers to preserve protein-protein interactions

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying phosphorylation states

• Binding conditions:

  • Optimize antibody-to-bead ratio

  • Consider pre-clearing lysates to reduce non-specific binding

  • Allow sufficient incubation time (typically overnight at 4°C)

• Washing stringency:

  • Balance between removing non-specific binding and preserving specific interactions

  • Consider detergent concentration and salt concentration in wash buffers

Research has used ERCC6 immunoprecipitation to identify interaction partners involved in DNA repair pathways, including the recent discovery of interactions between ERCC6 and histone chaperones like NAP1 in plant models .

How can ERCC6 antibodies be used to investigate the connection between chromatin remodeling and DNA repair mechanisms?

Recent research has identified ERCC6 as a chromatin remodeler involved in DNA repair processes. Advanced methodological approaches using ERCC6 antibodies include:

• Chromatin immunoprecipitation (ChIP):

  • Use ERCC6 antibodies to identify genomic regions where ERCC6 is recruited

  • Compare binding patterns before and after DNA damage induction

  • Recent plant research has employed this approach to study ERCC6's role in base excision repair

• Protein interaction studies:

  • Co-immunoprecipitation with ERCC6 antibodies to identify interaction partners

  • Recent research identified interactions between ERCC6 and histone chaperones like NAP1

  • Data showed that the catalytic ATPase domain of ERCC6 facilitates interactions with both AP endonuclease-redox protein (ARP) and NAP1

• Functional chromatin studies:

  • Investigate how ERCC6 and its partners affect nucleosome dynamics

  • Recent findings demonstrated that ERCC6 and NAP1 synergistically contribute to nucleosome sliding and exposure of hindered endonuclease cleavage sites

  • Loss-of-function mutations in ERCC6 resulted in hypersensitivity to DNA-damaging agents that induce base excision repair

These approaches have revealed that similar protein interactions are found across different species, suggesting a conserved recruitment mechanism employed by AP endonuclease to overcome chromatin barriers during DNA repair progression .

What role does ERCC6 play in genetic pleiotropy and multi-system disorders?

ERCC6 mutations have been associated with multiple disorders affecting different biological systems. Research approaches using ERCC6 antibodies to investigate this pleiotropy include:

• Large-scale biobank studies:

  • Recent meta-analysis of results from the BioMe Biobank and UK Biobank showed significant associations between deleterious ERCC6 variants and multiple disorders

  • Data showed odds ratios of 2.6 for retinal dystrophy (95% CI 1.5–4.6; P=8.7 × 10⁻⁴), 3.5 for atypical atrial flutter (95% CI 1.9–6.5; P=6.2 × 10⁻⁵), 1.5 for arrhythmia (95% CI 1.2–2.0; P=2.7 × 10⁻³), and 3.8 for lymphocyte immunodeficiency (95% CI 2.1–6.8; P=5.0 × 10⁻⁶)

• Phenotypic characterization:

  • ERCC6 antibodies can be used to study protein expression in different tissues

  • Recent findings showed carriers of ERCC6 loss-of-function variants who lacked formal diagnoses of these conditions still exhibited increased symptoms, indicating potential underdiagnosis

• Functional validation:

  • Western blot with ERCC6 antibodies can confirm the effects of mutations on protein expression

  • Recent studies on Cockayne syndrome patients demonstrated the deleterious effect of a novel ERCC6 mutation through Western blot analysis

These studies have revealed a unique genetic link among retinal, cardiac, and immune disorders, underscoring the value of EHR-linked biobanks in assessing the full clinical profile of carriers of rare ERCC6 variants .

How are ERCC6 antibodies used in studying Cockayne syndrome and related disorders?

Cockayne syndrome (CS) is a rare autosomal recessive disorder characterized by growth failure, neurological abnormalities, and photosensitivity. Methodological approaches using ERCC6 antibodies include:

• Mutation characterization:

  • Western blot with ERCC6 antibodies can demonstrate the effects of mutations on protein expression

  • Recent research described a novel ERCC6 mutation in three CS patients from independent families originating from northwestern Tunisia

  • The deleterious effect on the CSB protein was confirmed by Western blot analysis

• Functional assays:

  • UV sensitivity assays incorporating ERCC6 antibody detection

  • Recent studies showed defective repair of UV-induced DNA damage in CS patient cells

  • ERCC6 antibodies help confirm knockdown or knockout efficiency in cellular models

• Genotype-phenotype correlations:

  • Compare ERCC6 protein levels and function across patients with different mutations

  • Correlate molecular findings with clinical phenotypes

  • Recent studies have noted remarkable phenotypic differences even among siblings carrying the same mutations

These approaches have provided insights into the molecular basis of CS and related disorders, potentially identifying therapeutic targets and improving diagnostic approaches for these rare but severe conditions.

What approaches using ERCC6 antibodies can help investigate its role in age-related macular degeneration?

Genetic variation in ERCC6 has been associated with susceptibility to age-related macular degeneration type 5 (ARMD5) . Research methodologies using ERCC6 antibodies include:

• Expression analysis in retinal tissues:

  • Immunohistochemistry with ERCC6 antibodies can map expression patterns in retinal layers

  • Compare ERCC6 localization and levels between healthy and AMD-affected tissues

  • Correlate with disease progression and severity

• Variant characterization:

  • Express wild-type and variant ERCC6 in cell models

  • Use Western blot with ERCC6 antibodies to assess differences in protein expression and stability

  • Recent large-scale studies found a burden of deleterious variants in ERCC6 strongly associated with retinal disorders (OR=2.6, 95% CI 1.5–4.6; P=8.7 × 10⁻⁴)

• DNA damage response in retinal cells:

  • Track ERCC6 recruitment to damage sites using immunofluorescence

  • Compare response in cells with wild-type versus variant ERCC6

  • Correlate with DNA repair efficiency and cell survival

These approaches can help elucidate the mechanisms by which ERCC6 variants contribute to AMD pathogenesis and potentially identify new therapeutic strategies for this common cause of vision loss in elderly populations.