ERCC2 Antibody

What is ERCC2 and why are ERCC2 antibodies important research tools?

ERCC2 (Excision Repair Cross-Complementing Rodent Repair Deficiency, Complementation Group 2) functions as a DNA repair gene encoding an ATP-dependent 5'-3' helicase. It forms a critical component of the transcription factor II Human (TFIIH) complex, which plays essential roles in both nucleotide excision repair (NER) and basal transcription . ERCC2 is also known as XPD and is involved in separating the double helix during DNA repair processes . Mutations in ERCC2 can cause various diseases including trichothiodystrophy-1 (TTD1), xeroderma pigmentosum (XP), and XP/CS combinations with varying clinical severity .

ERCC2 antibodies serve as invaluable tools for investigating DNA repair mechanisms, cancer biomarkers, and immunological dysfunction. ERCC2 variants have been observed in multiple cancers and can serve as biomarkers to predict response to neoadjuvant treatment, particularly cisplatin-based chemotherapy in muscle-invasive bladder cancer . Recent research has also revealed ERCC2's unexpected role in B-cell function, with deficiencies linked to antibody production impairment and immunodeficiency .

Selecting the appropriate ERCC2 antibody requires consideration of several factors to ensure optimal experimental results:

• Target epitope: Different ERCC2 antibodies target distinct regions of the protein. N-terminal antibodies (such as ABIN2792558) recognize the amino-terminal region with the sequence KLNVDGLLVY FPYDYIYPEQ FSYMRELKRT LDAKGHGVLE MPSGTGKTVS . Consider whether your experiment requires detection of specific domains or if certain regions might be masked in protein complexes.

• Clonality and host species: Both polyclonal (e.g., rabbit polyclonal) and monoclonal ERCC2 antibodies are available . Polyclonal antibodies may provide higher sensitivity by recognizing multiple epitopes but potentially lower specificity, while monoclonal antibodies offer higher specificity for a single epitope. Consider the host species (typically rabbit) in relation to your experimental system to avoid cross-reactivity issues.

• Species cross-reactivity: Verify the antibody's reactivity with your species of interest. Many ERCC2 antibodies show reactivity with human samples primarily, with cited reactivity in mouse and rat models . Some antibodies (like ABIN2792558) report broader cross-reactivity including cow, guinea pig, and zebrafish .

• Validation status: Prioritize antibodies with published validation data in applications similar to your intended use. The number of publications citing an antibody can provide confidence in its reliability .

• Purification method: Affinity-purified antibodies typically offer higher specificity. Consider the immunogen used to generate the antibody (synthetic peptide vs. recombinant protein) as this affects epitope recognition .

Prior to commencing major experiments, perform validation tests with positive controls (cell lines known to express ERCC2 such as HeLa or HEK-293) and negative controls (ERCC2 knockdown cells if available) to confirm antibody specificity in your experimental system.

What is the optimal protocol for Western blot detection of ERCC2?

Based on published methodology, the following optimized protocol is recommended for Western blot detection of ERCC2:

Sample Preparation:

• Harvest cells and resuspend in lysis buffer

• Incubate on ice and centrifuge to collect supernatant

• Determine protein concentration using DC Protein Assay or equivalent

• Prepare samples containing 20 μg total protein in Laemmli buffer

Electrophoresis and Transfer:

• Load samples on NuPAGE 4-12% Bis-Tris gels

• Run electrophoresis using MOPS SDS running buffer

• Transfer to nitrocellulose membrane using NuPage Transfer Buffer (1×) with 20% ethanol and antioxidants

• Block membrane with milk powder in TBS + 0.05% Tween 20

Antibody Incubation and Detection:

• Incubate with rabbit anti-human ERCC2 (XPD) polyclonal antibody at 1 μg/ml overnight at 4°C

• Wash membrane thoroughly with TBS-T (3-5 washes, 5 minutes each)

• Incubate with HRP-conjugated secondary antibody

• Develop using chemiluminescent substrate and image with a chemiluminescence detector

• Include GAPDH (100 ng/ml antibody) as loading control

This protocol has been successfully used to detect ERCC2 in lymphoblastoid cell lines from trichothiodystrophy-1 patients with ERCC2 mutations, enabling comparison between mutant and wild-type protein expression . The expected molecular weight for ERCC2 is approximately 80 kDa in most gel systems, though the calculated molecular weight is 87 kDa .

How can ERCC2 antibodies be applied in research on B-cell function and immunodeficiency?

ERCC2 antibodies serve as essential tools for investigating the unexpected role of ERCC2 in B-cell function and immunodeficiency. Recent research has revealed that ERCC2 deficiency leads to antibody deficiency, with most TTD1 patients harboring ERCC2 mutations presenting with low serum IgG levels . The following methodological approaches incorporate ERCC2 antibodies:

• Protein expression analysis: Western blot analysis using ERCC2 antibodies enables quantification of ERCC2 protein levels in patient-derived cells. For TTD1 patients with compound heterozygous ERCC2 mutations, reduced or absent ERCC2 protein can be demonstrated using rabbit anti-human ERCC2 polyclonal antibody (1 μg/ml), with GAPDH (100 ng/ml) serving as loading control .

• B-cell activation studies: After isolating naïve CD19+ B-cells and stimulating them via the B-cell receptor (BCR), researchers can examine activation marker expression (CD69, CD86) in relation to ERCC2 expression. Impaired upregulation of these markers has been observed in ERCC2-deficient B-cells, suggesting defective BCR-mediated activation .

• Proliferation assays: ERCC2 antibodies can help characterize the relationship between ERCC2 expression and lymphocyte proliferation capacity following stimulation with pokeweed mitogen (PWM) or other activators .

• DNA repair assessment: Combining UV-irradiation assays with ERCC2 antibody detection enables correlation of DNA repair efficiency with protein expression levels in ERCC2-mutant cells.

• Immunophenotyping correlation: Flow cytometry data on B-cell subpopulations can be correlated with ERCC2 expression levels to understand the relationship between protein function and cellular phenotype.

These methodologies have helped establish that ERCC2 deficiency contributes to impaired B-cell activation and differentiation, resulting in antibody deficiency and increased susceptibility to infections .

What approaches can be used to study the effect of ERCC2 knockdown or mutation in cancer cells?

Multiple complementary approaches utilizing ERCC2 antibodies can be employed to investigate ERCC2's role in cancer biology:

• siRNA-mediated knockdown validation: Multiple siRNAs targeting different regions of ERCC2 (e.g., si-ERCC2-1, si-ERCC2-2, si-ERCC2-3) can be used to reduce ERCC2 expression, with knockdown efficiency confirmed by Western blot using ERCC2 antibodies .

• Proliferation assays: After confirming ERCC2 knockdown, researchers can assess cancer cell proliferation using multiple methods:

  • CCK-8 assay measuring proliferation at 24, 48, 72, and 96 hours

  • Colony formation assay quantifying the number and size of colonies formed

  • Three-dimensional multicellular sphere assays monitoring growth and morphology

  • Live/dead cell analysis using calcein-AM/PI co-staining

• Migration and invasion assays: ERCC2 knockdown effects on cancer cell motility can be evaluated through:

  • Wound healing assays tracking wound closure over time

  • Transwell assays quantifying cell migration and invasion capacities

• Nucleotide excision repair (NER) capacity assessment: Microscopy-based NER assays can evaluate how specific ERCC2 mutations affect DNA repair function, with ERCC2 antibodies enabling visualization of the protein's localization and interaction with repair complexes .

• Cisplatin sensitivity testing: Introducing ERCC2 mutations into bladder cancer cell lines, followed by cisplatin treatment, can demonstrate how specific mutations drive chemosensitivity in orthotopic xenograft models .

Research using these approaches has revealed that ERCC2 knockdown significantly inhibits proliferation, migration, and invasion of bladder cancer cells (T24 cell line) . Additionally, most ERCC2 helicase domain mutations impair NER capacity, which correlates with increased cisplatin sensitivity in muscle-invasive bladder cancer .

How can ERCC2 antibodies contribute to developing predictive biomarkers for cancer therapy response?

ERCC2 antibodies play a crucial role in developing and validating predictive biomarkers for cancer therapy response, particularly for cisplatin-based chemotherapy in muscle-invasive bladder cancer. The following methodological approaches leverage ERCC2 antibodies for biomarker development:

• Functional assessment of mutation impact: A microscopy-based NER assay, utilizing ERCC2 antibodies, can functionally classify clinically observed ERCC2 mutations. This approach has demonstrated that most ERCC2 helicase domain mutations cannot support NER, correlating with increased cisplatin sensitivity .

• Combined genomic and functional profiling: ERCC2 antibodies enable correlation between genomic mutation status and functional protein expression/activity. This combined approach provides superior predictive power compared to genomic analysis alone, potentially guiding therapy decisions in bladder cancer and other malignancies .

• Immunohistochemical scoring systems: ERCC2 antibody-based IHC can be standardized to develop scoring systems correlating protein expression patterns with treatment response across tumor samples.

• Cell line model validation: ERCC2 antibodies confirm the creation of ERCC2-deficient cancer models for studying treatment response. Research has shown that introducing ERCC2 mutations into bladder cancer cell lines abrogates NER activity and drives cisplatin sensitivity in xenograft models .

• Prospective clinical application: Institution-wide tumor profiling initiatives are incorporating ERCC2 mutation analysis and functional assessment using ERCC2 antibodies to guide treatment decisions .

These approaches support the direct role of ERCC2 mutations in determining cisplatin response and demonstrate how functional characterization of ERCC2 using antibody-based techniques provides clinically relevant information beyond genomic sequencing alone.

What methods exist for validating novel ERCC2 mutations and establishing their functional consequences?

Validating novel ERCC2 mutations and determining their functional significance requires a multi-faceted approach where ERCC2 antibodies play a central role:

• Protein expression analysis: Western blotting with ERCC2 antibodies can determine if mutations affect protein expression levels or stability. For TTD1 patients with compound heterozygous ERCC2 mutations, reduced or absent protein expression has been demonstrated using rabbit anti-human ERCC2 polyclonal antibody compared to healthy controls .

• Microscopy-based NER assays: These assays assess the functional impact of ERCC2 mutations on nucleotide excision repair capacity. ERCC2 antibodies enable visualization of the protein's localization and interaction with repair complexes, revealing that most helicase domain mutations cannot support NER activity .

• UV sensitivity testing: Cells harboring ERCC2 mutations typically show increased sensitivity to UV irradiation due to impaired DNA repair. ERCC2 antibodies can confirm the mutation's effect on protein expression in these functional assays .

• B-cell function assessment: For mutations affecting immune function, researchers can isolate naïve CD19+ B-cells from patients, stimulate them via BCR activation, and assess activation marker (CD69, CD86) expression in relation to ERCC2 levels. This approach has demonstrated that ERCC2 deficiency leads to impaired BCR-mediated B-cell activation .

• Complementation studies: Introducing wild-type ERCC2 into mutant cells should restore normal function if the mutation is causative. ERCC2 antibodies can confirm successful expression of the introduced wild-type protein.

• In silico structural analysis: Computational approaches can predict how specific mutations might affect protein structure and function, with antibody-based techniques validating these predictions experimentally.

These methodologies have successfully validated pathogenic ERCC2 mutations in patients with trichothiodystrophy-1 and established their role in causing DNA repair deficiency and impaired immune function .

How can researchers investigate interactions between ERCC2 and the TFIIH complex?

Investigating interactions between ERCC2 and the TFIIH complex requires specialized techniques where ERCC2 antibodies serve as essential tools:

• Co-immunoprecipitation (Co-IP): ERCC2 antibodies can be used to immunoprecipitate ERCC2 along with its interacting partners from the TFIIH complex. This approach typically employs 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate, followed by Western blot analysis of precipitated proteins .

• Reciprocal Co-IP: Antibodies against other TFIIH components can be used for immunoprecipitation, followed by Western blot with ERCC2 antibodies to confirm interaction.

• Proximity ligation assays (PLA): This technique visualizes protein-protein interactions in situ using primary antibodies against ERCC2 and other TFIIH components, followed by secondary antibodies conjugated to oligonucleotides that produce fluorescent signals when proteins are in close proximity.

• Immunofluorescence co-localization: ERCC2 antibodies combined with antibodies against other TFIIH components can demonstrate co-localization at sites of DNA repair or transcription.

• Chromatin immunoprecipitation (ChIP): ERCC2 antibodies can be used in ChIP assays to identify DNA sequences associated with ERCC2 and the TFIIH complex during transcription or repair processes.

• Mass spectrometry analysis: Following immunoprecipitation with ERCC2 antibodies, mass spectrometry can identify all interacting partners, potentially revealing novel interactions beyond known TFIIH components.

These methodologies help understand how mutations in ERCC2 affect its interaction with the TFIIH complex, potentially explaining the diverse phenotypes observed in patients with different ERCC2 mutations, including cancer susceptibility, DNA repair deficiency, and immune dysfunction .

What are common technical challenges when working with ERCC2 antibodies and how can they be addressed?

Researchers working with ERCC2 antibodies frequently encounter several technical challenges that can be addressed through specific optimization strategies:

• Weak signal intensity in Western blot

  • Challenge: Nuclear proteins like ERCC2 may be present at relatively low abundance.

  • Solution: Use optimized nuclear extraction protocols; increase protein loading to 20-30 μg; extend primary antibody incubation to overnight at 4°C; use recommended antibody dilutions (1:500-1:1000); employ enhanced chemiluminescence detection systems .

• Multiple bands or high background

  • Challenge: Nonspecific binding can complicate interpretation of results.

  • Solution: Increase blocking stringency using 5% milk or BSA in TBS + 0.05% Tween 20; optimize antibody dilution; increase wash frequency and duration; validate with positive controls (HeLa, K-562, HEK-293 cells) and negative controls (ERCC2 knockdown) .

• Discrepancy between expected and observed molecular weight

  • Challenge: ERCC2 runs at approximately 80 kDa despite a calculated molecular weight of 87 kDa.

  • Solution: Use protein ladders with clear markers; include positive control lysates; be aware of this established discrepancy when interpreting results .

• Variability in immunohistochemistry staining

  • Challenge: Inconsistent staining patterns or intensity across samples.

  • Solution: Standardize fixation protocols; use recommended antigen retrieval with TE buffer pH 9.0 (or alternatively citrate buffer pH 6.0); optimize antibody dilution within the recommended range (1:20-1:200); include positive control tissues (human lymphoma or cervical cancer tissue) .

• Poor reproducibility between experiments

  • Challenge: Results vary significantly between replicates.

  • Solution: Maintain consistent sample preparation protocols; use the same antibody lot when possible; implement detailed documentation procedures; include appropriate controls in each experiment .

• Optimizing for multi-antibody techniques

  • Challenge: Techniques requiring multiple antibodies (e.g., co-localization) present compatibility issues.

  • Solution: Select antibodies from different host species to avoid cross-reactivity; validate antibody combinations on control samples before proceeding to experimental conditions.

These optimization strategies have been successfully implemented in studies investigating ERCC2's role in DNA repair, cancer biology, and immune function .

How should researchers design experiments to investigate ERCC2 mutations in patient samples?

Designing robust experiments to investigate ERCC2 mutations in patient samples requires careful planning and methodological consideration:

• Comprehensive sample collection and processing

  • Collect peripheral blood for isolation of peripheral blood mononuclear cells (PBMCs)

  • Generate lymphoblastoid cell lines (LCLs) from patient samples for sustained experimentation

  • Obtain appropriate control samples from healthy individuals matched for age and sex

  • Process all samples using standardized protocols to ensure comparability

• Mutation verification and characterization

  • Confirm mutations through DNA sequencing

  • Use in silico tools to predict functional consequences

  • Compare novel mutations with established pathogenic variants

  • Document clinical features to correlate with genotype

• Protein expression analysis

  • Prepare lysates containing 20 μg total protein

  • Run Western blots using rabbit anti-human ERCC2 antibody (1 μg/ml)

  • Include GAPDH (100 ng/ml) as loading control

  • Compare expression levels between patient samples and controls

• Functional assays for DNA repair capacity

  • UV irradiation assays to assess DNA repair efficiency

  • Microscopy-based NER assays to directly evaluate repair capacity

  • Compare repair capacity between patient cells and controls

• Cell-type specific investigation

  • For immunological studies, isolate specific cell populations (e.g., naïve CD19+ B-cells)

  • Perform activation assays (e.g., BCR stimulation with anti-IgM in presence of IL-2)

  • Measure activation markers (CD69, CD86) after 24 hours by flow cytometry

  • Compare responses between patient cells and healthy controls

• Gene expression profiling

  • Perform RNA sequencing on patient-derived cells

  • Compare transcriptional profiles with healthy controls

  • Identify dysregulated pathways and genes

  • Validate key findings by qPCR or protein-level analysis

This comprehensive approach has successfully identified the functional consequences of novel ERCC2 mutations, including impaired DNA repair and B-cell dysfunction, establishing ERCC2 as an important gene in both cancer biology and immunology .

How are ERCC2 antibodies being applied in emerging cancer biomarker research?

ERCC2 antibodies are increasingly being utilized in novel cancer biomarker research approaches that extend beyond traditional applications:

• Combined genomic-functional biomarker development: Researchers are developing approaches that integrate ERCC2 mutation status with protein expression and functional assessment. ERCC2 antibodies enable microscopy-based NER assays that can functionally classify ERCC2 mutations according to their impact on DNA repair capacity, providing superior predictive power for cisplatin response in muscle-invasive bladder cancer .

• Multi-cancer application: While initial biomarker development focused on bladder cancer, research is expanding to investigate ERCC2's role in other malignancies. ERCC2 antibodies are being employed to examine protein expression patterns across cancer types, potentially identifying additional cancer subtypes that might benefit from specific treatment approaches .

• Immunotherapy response prediction: Given ERCC2's emerging role in immune function, researchers are exploring whether ERCC2 status might predict response to immunotherapies. ERCC2 antibodies help characterize the relationship between DNA repair capacity and immune response in the tumor microenvironment .

• Dynamic biomarker monitoring: Rather than static assessment, researchers are investigating changes in ERCC2 expression during treatment as a dynamic biomarker. ERCC2 antibodies enable monitoring of protein levels in sequential samples, potentially capturing treatment-induced changes that might predict resistance development .

• Multi-parameter predictive models: ERCC2 antibody-based assays are being incorporated into comprehensive predictive models that integrate multiple biomarkers. These approaches aim to improve prediction accuracy by considering ERCC2 alongside other DNA repair proteins and signaling pathway components .

These emerging applications highlight ERCC2's potential as both a predictive biomarker for treatment response and a target for therapeutic development, with ERCC2 antibodies playing a central role in advancing this research.

What novel roles for ERCC2 are being discovered through antibody-based research?

Antibody-based research is uncovering unexpected roles for ERCC2 beyond its established functions in DNA repair and transcription:

• B-cell activation and antibody production: Recent research using ERCC2 antibodies has revealed ERCC2's critical role in B-cell function. Studies in trichothiodystrophy-1 patients with ERCC2 mutations demonstrated impaired BCR-mediated B-cell activation and antibody deficiency. Specifically, naïve CD19+ B-cells from ERCC2-deficient patients showed decreased upregulation of CD69 and CD86 after BCR stimulation, indicating ERCC2's unexpected importance in immune function .

• Cancer cell proliferation and metastasis: ERCC2 knockdown studies in bladder cancer cells, validated using ERCC2 antibodies, demonstrated that loss of ERCC2 function significantly inhibits proliferation, migration, and invasion capacities. These findings suggest ERCC2 actively contributes to cancer progression beyond its role in treatment response .

• Transcriptional regulation during immune responses: Differential gene expression analysis in ERCC2-deficient cells has revealed that ERCC2 influences the transcription of genes involved in growth factor signaling and B-cell activation. ERCC2 antibodies help establish the mechanistic link between mutation status and transcriptional dysregulation .

• Potential role in extracellular vesicle research: The ERCC2 repository has published findings related to extracellular vesicles (EVs) and exRNA research, suggesting potential connections between ERCC2 and intercellular communication mechanisms that merit further investigation .

• Interactions with immune checkpoint regulation: Emerging research is exploring correlations between ERCC2 expression and immune checkpoint molecules, with ERCC2 antibodies enabling investigation of potential mechanistic connections .

These discoveries highlight how antibody-based research continues to expand our understanding of ERCC2's biological roles, with implications extending from cancer therapy to immunological disorders and beyond.