ERCC1 Antibody, HRP conjugated

What are the recommended applications for ERCC1 Antibody, HRP conjugated?

ERCC1 Antibody with HRP conjugation can be effectively utilized across multiple laboratory techniques. Based on validated protocols, the primary applications include Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), and Flow Cytometry (FC). For Western Blotting applications, a dilution range of 1:500 to 1:2000 is typically recommended, while IHC protocols generally utilize a 1:150 dilution. For Immunofluorescence and Flow Cytometry, a 1:100 dilution has been established as optimal for most experimental setups . When planning experiments, it's essential to validate these dilutions in your specific experimental system as sensitivity may vary between tissue types and experimental conditions.

What are the optimal storage conditions for maintaining ERCC1 Antibody, HRP conjugated activity?

Proper storage is critical for maintaining antibody functionality and preventing degradation of the HRP conjugate. ERCC1 Antibody with HRP conjugation should be stored at -20°C in its original formulation upon receipt . The antibody is typically supplied in a buffer containing stabilizers such as 50% glycerol or similar cryoprotectants to maintain stability during freeze-thaw cycles . When working with the antibody:

• Aliquot the stock solution into smaller volumes to minimize freeze-thaw cycles

• Avoid exposure to light as HRP conjugates are photosensitive

• Minimize repeated freeze-thaw cycles which can progressively reduce antibody activity

• Allow the antibody to equilibrate to room temperature before opening to prevent condensation

Under these conditions, the antibody generally maintains stability for approximately 12 months from the date of receipt .

How can I optimize Western blotting protocols when using ERCC1 Antibody, HRP conjugated?

Optimization of Western blotting protocols for ERCC1 detection requires attention to several critical factors:

• Sample preparation: ERCC1 has a molecular weight of approximately 39 kDa , so ensure your gel percentage accommodates optimal resolution in this range.

• Protein loading: Since ERCC1 may be expressed at variable levels depending on the sample type, start with 20-50 μg of total protein lysate.

• Blocking optimization: Use 3-5% BSA or milk in TBST as blocking agent, testing both to determine which provides better signal-to-noise ratio.

• Antibody dilution: Begin with the recommended 1:1000 dilution for Western blotting, and adjust based on signal intensity.

• Incubation conditions: Incubate with primary antibody overnight at 4°C with gentle rocking to improve specific binding.

• Detection method: Since the antibody is HRP-conjugated, use an appropriate chemiluminescent substrate with sensitivity matched to your expected expression level.

• Exposure optimization: Capture multiple exposure times to ensure linear range detection of your protein of interest.

A methodological approach is to always include appropriate positive controls, such as cell lines known to express ERCC1 (e.g., certain lung cancer or ovarian cancer cell lines), alongside experimental samples to validate antibody performance in each experiment.

How can ERCC1 antibodies be used to evaluate chemoresistance in cancer research?

ERCC1 expression levels have been established as potential biomarkers for predicting resistance to platinum-based therapies in various cancer types. When designing experiments to investigate chemoresistance:

• Baseline expression analysis: Establish ERCC1 expression levels across cell lines or patient samples using the HRP-conjugated antibody in immunohistochemistry or Western blotting applications.

• Correlation studies: Compare ERCC1 expression with clinical outcomes or in vitro drug sensitivity. Research has demonstrated that high ERCC1 expression correlates with resistance to platinum agents in gastric and non-small cell lung cancers .

• Isoform-specific detection: Consider that different ERCC1 isoforms may contribute differently to cisplatin resistance. While isoform 202 has been specifically linked to cisplatin resistance in lung cancer models , an antibody detecting multiple isoforms may provide more comprehensive information about potential resistance mechanisms.

• Functional validation: Combine expression studies with functional assays, such as knockdown or overexpression of ERCC1, followed by drug sensitivity testing.

• Combined biomarker approach: Assess ERCC1 in conjunction with other DNA repair proteins such as XPF, as research has shown their coordinated expression may provide more predictive power regarding treatment response .

When interpreting results, consider that while ERCC1 overexpression is generally associated with chemoresistance, the specific threshold defining "high" versus "low" expression may vary across tumor types and experimental systems.

What are the considerations for detecting different ERCC1 isoforms using antibodies?

The ERCC1 gene produces four main isoforms (201, 202, 203, and 204) through alternative splicing, with potentially different functional relevance to DNA repair and chemoresistance . When selecting antibodies for isoform analysis:

Research indicates that antibodies generated against the full-length ERCC1 protein may detect multiple isoforms, while those raised against specific peptide sequences may offer isoform selectivity .

How should I approach cross-reactivity testing when using ERCC1 antibodies across different species?

When planning experiments involving samples from different species, cross-reactivity assessment is crucial for generating reliable data. For ERCC1 antibodies:

• Sequence homology analysis: Review the antibody specificity information provided by manufacturers. For example, some ERCC1 antibodies share 100% sequence homology with human, mouse, rat, hamster, and monkey antigens .

• Validation strategy:

  • Begin with positive control samples from each species of interest

  • Compare staining patterns and band sizes across species

  • Include appropriate negative controls (e.g., ERCC1 knockout samples if available)

  • Validate specificity using immunoprecipitation followed by mass spectrometry

• Empirical testing protocol:

  • For Western blotting: Run samples from multiple species side-by-side using identical conditions

  • For IHC/IF: Test antibody performance on fixed tissue sections from different species using the same staining protocol

  • Adjust antibody concentrations for each species based on signal-to-noise ratio

• Documentation of species reactivity: Carefully document confirmed reactivity, as manufacturers may list theoretical cross-reactivity based on sequence homology rather than experimental validation .

When interpreting cross-species results, be aware that differences in post-translational modifications between species may affect antibody binding even when the primary sequence is conserved.

What are common causes of background or non-specific binding when using ERCC1 Antibody, HRP conjugated, and how can they be addressed?

High background or non-specific binding can compromise data quality when using ERCC1 antibodies with HRP conjugation. Common causes and solutions include:

• Suboptimal blocking:

  • Problem: Insufficient blocking allows non-specific antibody binding

  • Solution: Optimize blocking by testing different agents (BSA vs. milk) and concentrations (3-5%)

  • Extend blocking time to 1-2 hours at room temperature

• Excessive antibody concentration:

  • Problem: Too concentrated antibody solutions increase non-specific binding

  • Solution: Titrate antibody concentration, starting with manufacturer's recommendation (e.g., 1:1000 for WB , 1:150 for IHC )

  • Consider longer incubation times with more dilute antibody solutions

• Inadequate washing:

  • Problem: Residual unbound antibody contributes to background

  • Solution: Increase wash duration and frequency (4-5 washes, 5-10 minutes each)

  • Use gentle agitation during washing steps

• Sample preparation issues:

  • Problem: Endogenous peroxidase activity in tissues or cells

  • Solution: Include hydrogen peroxide quenching step (0.3% H₂O₂ in methanol for 15-30 minutes) before antibody incubation for IHC applications

• Detection system sensitivity:

  • Problem: Overly sensitive detection reagents amplify background

  • Solution: Adjust substrate incubation time or switch to less sensitive detection reagents

  • Capture images at multiple exposure times to determine optimal signal-to-noise ratio

Maintain a systematic troubleshooting approach by changing only one variable at a time and documenting outcomes to identify the optimal protocol for your specific experimental system.

How can I validate the specificity of ERCC1 antibody detection in my experimental system?

Rigorous validation of antibody specificity is essential for generating reproducible and reliable research data. For ERCC1 antibody validation:

• Positive and negative control samples:

  • Use cell lines with known high (e.g., certain cisplatin-resistant cancer lines) and low ERCC1 expression

  • Include ERCC1 knockout or knockdown samples as negative controls

  • Test tissues with established ERCC1 expression patterns

• Molecular weight verification:

  • Confirm that the detected band appears at the expected molecular weight (~39 kDa)

  • Be aware of potential post-translational modifications that may alter apparent molecular weight

• Orthogonal detection methods:

  • Compare antibody-based detection with mRNA expression data

  • Verify results using alternative ERCC1 antibodies targeting different epitopes

  • Confirm specificity using mass spectrometry following immunoprecipitation

• Functional validation:

  • Correlate ERCC1 expression with known biological functions (e.g., DNA repair capacity or cisplatin resistance)

  • Perform rescue experiments by reintroducing ERCC1 in knockout models

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide to block specific binding

  • Compare staining patterns between blocked and unblocked antibody conditions

Each validation approach contributes complementary evidence supporting antibody specificity, and results should be documented thoroughly to support publication requirements for antibody validation.

What factors might affect the detection of ERCC1 in tissue samples using HRP-conjugated antibodies?

Multiple factors can influence ERCC1 detection in tissue samples, potentially leading to variability in experimental results:

• Fixation parameters:

  • Fixation method: Formalin fixation may mask epitopes compared to frozen sections

  • Fixation duration: Overfixation can reduce antibody accessibility to epitopes

  • Solution: Optimize antigen retrieval methods (heat-induced vs. enzymatic) for formalin-fixed samples

• Sample age and storage:

  • Problem: Antigen degradation in archived samples

  • Consideration: Interpret results cautiously when comparing fresh vs. archived samples

  • Control: Include standardized positive controls with each batch of staining

• Tissue-specific expression patterns:

  • Heterogeneous expression: ERCC1 expression varies across different cell types within the same tissue

  • Solution: Use higher magnification imaging to assess cell-specific expression patterns

  • Control: Compare results with established expression patterns in literature

• Pre-analytical variables:

  • Ischemia time: Protein degradation begins immediately after sample collection

  • Processing protocols: Standardize time from collection to fixation

  • Storage conditions: Maintain consistent temperature and humidity for tissue blocks and slides

• Technical considerations:

  • Antibody penetration: Tissue thickness affects antibody access (optimize section thickness to 4-5 μm)

  • Detection system sensitivity: Match detection method to expression level

  • Counterstaining intensity: Excessive counterstaining may mask specific signals

Research has shown that ERCC1 expression in hepatocellular carcinoma tissues is significantly lower than in adjacent tissues , highlighting the importance of appropriate controls and standardized protocols when comparing different tissue types.

How does ERCC1 expression correlate with clinical outcomes in different cancer types, and what are the implications for antibody-based testing?

ERCC1 expression has emerged as a potential biomarker for treatment response and prognosis across multiple cancer types, with important implications for antibody-based testing methodologies:

• Cancer-specific correlations:

  • Non-small cell lung cancer: High ERCC1 expression correlates with resistance to platinum-based therapies and poorer outcomes .

  • Gastric cancer: ERCC1 overexpression is associated with resistance to cisplatin and 5-fluorouracil-based therapy .

  • Hepatocellular carcinoma: Lower ERCC1 expression has been associated with early relapse after surgery, hepatic capsular invasion, and microvascular invasion .

• Methodological considerations for clinical testing:

  • Standardization challenges: Different antibody clones and detection methodologies may yield varying results

  • Threshold determination: Optimal cutoff values for "high" versus "low" expression remain undefined across cancer types

  • Scoring systems: Develop and validate scoring systems that account for both staining intensity and percentage of positive cells

• Multimarker approaches:

  • ERCC1-XPF complex: Assess both ERCC1 and XPF expression, as their coordinated expression may provide more comprehensive predictive information

  • DNA repair pathway analysis: Combine ERCC1 testing with assessment of other DNA repair proteins for more robust prediction

• Antibody selection criteria for clinical applications:

  • Prioritize antibodies with validated specificity for functionally relevant isoforms

  • Consider antibodies that detect multiple isoforms for comprehensive assessment

  • Select clones with established performance in the specific application (IHC vs. Western blotting)

When designing studies to evaluate ERCC1 as a biomarker, researchers should standardize antibody selection, staining protocols, and scoring systems to enable meaningful cross-study comparisons and eventual clinical translation.

What are the considerations for multiplexing ERCC1 detection with other DNA repair proteins?

Multiplexed detection of ERCC1 alongside other DNA repair proteins can provide more comprehensive insights into repair pathway functionality and treatment response prediction. Key considerations include:

Studies have demonstrated that the combined assessment of ERCC1 and XPF expression in hepatocellular carcinoma provides more informative prognostic information than either marker alone , supporting the value of multiplexed approaches.

How can ERCC1 antibodies be utilized to investigate the role of DNA repair in response to novel therapeutics beyond platinum agents?

While ERCC1's role in platinum drug resistance is well-established, emerging research highlights its potential significance in response to other therapeutic modalities:

• PARP inhibitor sensitivity:

  • Experimental approach: Use ERCC1 antibodies to stratify cell lines or patient samples, then assess correlation with PARP inhibitor sensitivity

  • Mechanistic insight: Low ERCC1 expression has been reported to increase sensitivity of lung cancer cells to PARP inhibitors

  • Research design: Combine ERCC1 immunodetection with functional assays measuring synthetic lethality

• Radiotherapy response prediction:

  • Methodology: Assess ERCC1 expression in pre-treatment biopsies using immunohistochemistry with HRP-conjugated antibodies

  • Analysis approach: Correlate expression levels with radiation response metrics

  • Mechanistic studies: Investigate how ERCC1 expression changes during fractionated radiotherapy using serial biopsies

• Immunotherapy biomarker exploration:

  • Hypothesis testing: Investigate whether DNA repair deficiency (low ERCC1) correlates with tumor mutational burden and immunotherapy response

  • Tissue analysis: Perform multiplexed staining of ERCC1 alongside immune cell markers

  • Spatial analysis: Assess ERCC1 expression in relation to tumor-immune cell interfaces

• Combination therapy optimization:

  • Experimental design: Determine whether ERCC1 expression predicts synergistic effects between DNA-damaging agents and targeted therapies

  • Dynamic assessment: Monitor changes in ERCC1 expression during treatment using longitudinal sampling

  • Functional validation: Compare antibody-based expression data with functional DNA repair capacity assays

When designing such studies, researchers should consider both nuclear and cytoplasmic ERCC1 localization, as subcellular distribution may provide additional functional information beyond total expression levels.

What role does ERCC1 antibody detection play in understanding cancer heterogeneity and its impact on treatment response?

Cancer heterogeneity presents significant challenges for treatment strategies, and ERCC1 antibody-based detection can provide valuable insights into this complexity:

• Intratumoral heterogeneity assessment:

  • Methodology: Analyze multiple regions within a single tumor using ERCC1 immunohistochemistry

  • Quantification approach: Develop spatial mapping of ERCC1 expression across tumor sections

  • Correlation analysis: Relate ERCC1 expression patterns to histopathological features and treatment response

• Single-cell analysis techniques:

  • Flow cytometry application: Use HRP-conjugated or fluorescently labeled ERCC1 antibodies for flow cytometric analysis of cell suspensions

  • Imaging mass cytometry: Combine ERCC1 antibody detection with multiple markers for high-dimensional single-cell phenotyping

  • Data analysis: Apply clustering algorithms to identify distinct cell populations based on ERCC1 and other markers

• Evolution of ERCC1 expression during treatment:

  • Serial biopsy studies: Monitor changes in ERCC1 expression patterns before, during, and after treatment

  • Circulating tumor cell analysis: Develop protocols for ERCC1 detection in CTCs as liquid biopsy approach

  • Predictive modeling: Develop algorithms to predict treatment response based on dynamic ERCC1 expression changes

• Clinical translation considerations:

  • Sampling strategy optimization: Determine minimum sampling requirements to capture heterogeneity

  • Scoring system development: Create heterogeneity-aware scoring systems that account for variable expression

  • Treatment stratification: Investigate whether predominant or minority ERCC1 expression patterns better predict outcomes

Research in hepatocellular carcinoma has demonstrated variable ERCC1 expression patterns associated with specific pathological features , highlighting the potential of ERCC1 detection for characterizing tumor heterogeneity with clinical relevance.

How can ERCC1 antibodies be used in developing and validating companion diagnostics for precision oncology?

The development of companion diagnostics based on ERCC1 detection requires rigorous validation and standardization processes:

• Assay development considerations:

  • Antibody selection: Evaluate multiple antibody clones for optimal sensitivity and specificity

  • Detection platform selection: Compare IHC, ELISA, and other platforms for reproducibility and clinical applicability

  • Reference standard development: Establish calibration materials and positive/negative controls

• Analytical validation requirements:

  • Precision assessment: Evaluate intra-laboratory and inter-laboratory reproducibility

  • Sensitivity determination: Establish limits of detection and quantification

  • Specificity confirmation: Demonstrate antibody specificity through multiple orthogonal approaches

• Clinical validation approach:

  • Retrospective cohort studies: Analyze archived samples with known treatment outcomes

  • Prospective clinical trials: Incorporate ERCC1 testing in prospective treatment stratification studies

  • Statistical planning: Determine appropriate sample sizes for powering biomarker validation studies

• Regulatory considerations:

  • Documentation requirements: Maintain comprehensive records of antibody validation

  • Quality control procedures: Develop standard operating procedures for assay performance

  • Reference laboratory standardization: Establish proficiency testing programs for clinical laboratories

Successful companion diagnostic development would require collaboration between academic researchers, industry partners, and regulatory agencies to establish standardized testing approaches that can reliably guide treatment decisions based on ERCC1 expression status.