EPS8L3 Antibody, HRP conjugated

What is EPS8L3 and why is it important in cancer research?

EPS8L3 is a member of the epidermal growth factor receptor (EGFR) kinase substrate 8 (EPS8) family. It has gained significance in cancer research due to its overexpression in hepatocellular carcinoma (HCC) tissues compared to adjacent non-tumor tissues and its association with poor clinical prognosis . Both in vitro and in vivo experiments have demonstrated that EPS8L3 promotes proliferative ability by downregulating p21/p27 expression and enhances migratory and invasive abilities by upregulating matrix metalloproteinase-2 expression . Furthermore, EPS8L3 has been shown to affect the activation of the EGFR-ERK pathway by modulating EGFR dimerization and internalization .

How does EPS8L3 expression differ between normal and cancerous tissues?

Analysis of The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases reveals that EPS8L3 mRNA expression is significantly higher in HCC tumor tissues compared to normal liver tissues . This overexpression pattern is also observed in several other cancer types including cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), pancreatic adenocarcinoma (PAAD), and rectum adenocarcinoma (READ) . Quantitative RT-PCR results from 51 pairs of fresh HCC samples and 92 pairs of fresh intrahepatic cholangiocarcinoma (ICC) samples have confirmed these findings .

What is the molecular weight and structure of the EPS8L3 protein?

The EPS8L3 protein has a calculated molecular weight of approximately 67 kDa . The protein is recognized by specific antibodies that target unique epitopes within the EPS8L3 sequence. The immunogen used for antibody production is derived from specific sequences within the EPS8L3 protein structure .

What are the primary applications for EPS8L3 antibodies in cancer research?

EPS8L3 antibodies are primarily used in Western blotting (WB), immunohistochemistry (IHC), and immunofluorescence/immunocytochemistry (IF/ICC) applications . In HCC research, these antibodies have been instrumental in demonstrating the overexpression of EPS8L3 in tumor tissues compared to adjacent non-tumorous samples through Western blotting analysis and IHC staining using tissue microarrays . They are also valuable tools for investigating the role of EPS8L3 in cell proliferation, migration, and invasion through various functional assays.

How can EPS8L3 antibodies be used to study its association with cancer stem cell markers?

EPS8L3 has been found to be associated with liver cancer stem cells (LCSCs) that are positive for CD24, CD13, and EpCAM markers . Researchers can employ EPS8L3 antibodies in multiplex immunofluorescence assays to co-stain for these LCSC markers and EPS8L3, helping to elucidate the relationship between EPS8L3 expression and cancer stemness. Flow cytometry analysis using fluorescently-labeled EPS8L3 antibodies can also be used to quantify EPS8L3 expression in sorted cell populations based on stemness markers .

What dilutions are recommended for different experimental applications of EPS8L3 antibodies?

For optimal results with EPS8L3 antibodies, the following dilutions are typically recommended:

• Western blot (WB): 1:1000

• Immunohistochemistry (IHC): 1:50-1:200

• Immunofluorescence/Immunocytochemistry (IF/ICC): 1:100-1:500

What advantages do HRP-conjugated EPS8L3 antibodies offer over unconjugated versions?

HRP-conjugated EPS8L3 antibodies provide direct enzymatic detection capability, eliminating the need for secondary antibody incubation steps in procedures such as Western blotting, ELISA, and immunohistochemistry. This results in shorter protocols, reduced background signal (as fewer antibodies are used), and potentially greater sensitivity due to the direct coupling of the detection enzyme. Additionally, HRP conjugation allows for flexible detection methods including colorimetric, chemiluminescent, and chemifluorescent substrates depending on the experimental requirements.

What are the optimal conditions for preserving the activity of HRP-conjugated EPS8L3 antibodies?

HRP-conjugated antibodies require careful storage to maintain enzymatic activity. They should be stored at -20°C in a buffer containing stabilizers such as glycerol (typically 50%) and small amounts of preservatives like sodium azide (0.02%) . Repeated freeze-thaw cycles should be avoided as they can degrade both the antibody binding capacity and the HRP enzymatic activity. When handling these conjugates, it's advisable to aliquot the stock solution upon first thaw to minimize the number of freeze-thaw cycles. For short-term use, storage at 4°C (up to two weeks) is generally acceptable if the antibody contains appropriate preservatives.

How can researchers verify the specificity of HRP-conjugated EPS8L3 antibodies?

To verify specificity, researchers should include appropriate controls in their experiments:

• Positive controls: Known EPS8L3-expressing tissues or cell lines (e.g., Huh7 cells, which express significant levels of EPS8L3)

• Negative controls: Tissues or cells with EPS8L3 knockdown using siRNA or shRNA

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide should abolish specific signals

• Western blot validation: Verification that the antibody detects a single band at the expected molecular weight of 67 kDa

• Cross-reactivity testing: Confirming specificity across species if using the antibody in non-human samples

How can researchers address weak or absent signals when using EPS8L3 antibodies in Western blots?

When encountering weak or absent signals with EPS8L3 antibodies:

• Increase protein loading: EPS8L3 might be expressed at low levels in some cell types

• Optimize extraction method: Ensure proper protein extraction with protease inhibitors to prevent degradation

• Adjust antibody concentration: Try increasing primary antibody concentration or incubation time

• Use enhanced detection systems: Employ high-sensitivity chemiluminescent substrates for HRP detection

• Verify sample quality: Check for sample degradation with a housekeeping protein control

• Enhance transfer efficiency: For the 67 kDa EPS8L3 protein, ensure adequate transfer time and buffer conditions

• Check cell type relevance: Confirm EPS8L3 expression in your cell type, as expression can vary significantly between different cancer cell lines

What strategies can help minimize background when using HRP-conjugated EPS8L3 antibodies?

To reduce background with HRP-conjugated antibodies:

• Optimize blocking conditions: Use 5% nonfat milk or BSA in TBS-T for 1 hour at room temperature

• Increase washing steps: Add additional or longer wash steps with TBS-T

• Dilute antibody properly: Follow recommended dilutions (1:1000 for WB) or optimize for your specific conditions

• Add protein to antibody diluent: Include 1-3% blocking agent in antibody dilution buffer

• Filter antibody solutions: Remove aggregates that might cause non-specific binding

• Reduce substrate incubation time: Shorten exposure to HRP substrate to minimize background development

• Pre-adsorb antibody: For tissue applications, consider pre-adsorption with tissue powder

How can researchers troubleshoot inconsistent results in EPS8L3 detection across different experiments?

For consistent results when working with EPS8L3 antibodies:

• Standardize protein extraction methods: Use consistent lysis buffers (e.g., RIPA buffer containing protease inhibitors)

• Control for EPS8L3 expression variability: Expression can vary with cell density and growth conditions

• Maintain consistent antibody storage: Aliquot antibodies to avoid repeated freeze-thaw cycles

• Standardize incubation conditions: Keep temperature, time, and agitation consistent

• Use internal controls: Include standard positive controls in each experiment

• Maintain consistent transfer conditions: For Western blots, standardize transfer times and buffer compositions

• Document lot numbers: Track antibody lots as there can be lot-to-lot variation

How can EPS8L3 antibodies be used to investigate the EGFR-ERK pathway in cancer cells?

EPS8L3 has been demonstrated to affect the activation of the EGFR-ERK pathway by modulating EGFR dimerization and internalization . Researchers can design co-immunoprecipitation experiments using EPS8L3 antibodies to pull down protein complexes and investigate interactions with EGFR and other pathway components. Additionally, immunofluorescence microscopy with EPS8L3 antibodies can visualize co-localization with EGFR before and after EGF stimulation. For studying EGFR internalization specifically, researchers can employ flow cytometry analysis of surface EGFR levels following EGF stimulation in cells with normal or altered EPS8L3 expression . Phospho-specific antibodies against ERK can be used in conjunction with EPS8L3 modulation to assess downstream pathway activation.

What methods can be used to investigate the functional impact of EPS8L3 on cancer cell phenotypes?

To investigate EPS8L3's functional impact:

• Proliferation assays: CCK-8 assays and colony formation assays can be performed after EPS8L3 knockdown or overexpression

• Cell cycle analysis: Flow cytometry can assess changes in cell cycle distribution (G0/G1, S, G2/M phases) when EPS8L3 is modulated

• Migration and invasion assays: Transwell assays with or without Matrigel coating can evaluate the effect of EPS8L3 on cell motility

• Sphere formation assays: To assess self-renewal ability, especially in the context of cancer stem cell properties

• In vivo tumor models: Xenograft models using cells with modulated EPS8L3 can assess tumor growth, metastasis, and response to therapy

• Expression analysis of downstream targets: Western blotting to examine p21/p27 and MMP-2 levels after EPS8L3 modulation

How can researchers use EPS8L3 antibodies to explore its role in cancer stem cell maintenance?

Recent research has identified EPS8L3 as a key functional gene associated with triple LCSC marker-positive HCC cells (CD24+/CD13+/EpCAM+) . To explore this role:

• Multiplex immunofluorescence staining: Co-staining for EPS8L3 and LCSC markers can reveal spatial relationships and co-expression patterns

• FACS-based approaches: Sorting cells based on LCSC markers followed by EPS8L3 analysis or vice versa

• Functional assays: Sphere formation assays after EPS8L3 knockdown can assess impact on self-renewal capacity

• Transcriptional regulation studies: ChIP assays using antibodies against transcription factors like SP1 can investigate the regulation of EPS8L3 expression

• RNA-seq analysis: Compare transcriptomes of EPS8L3-high vs. EPS8L3-low cells within LCSC populations

• Pathway analysis: Identify signaling pathways connecting EPS8L3 with stemness maintenance

How should researchers interpret EPS8L3 expression data in relation to patient prognosis?

When analyzing EPS8L3 expression data:

What are the important considerations when analyzing EPS8L3 expression across different cancer types?

When comparing EPS8L3 expression across cancer types:

• Tissue-specific variation: Normal baseline expression of EPS8L3 varies between tissues, necessitating tissue-specific normalization

• Cancer subtype analysis: Expression patterns may differ across cancer subtypes (e.g., different HCC etiologies)

• Data integration: Combine data from multiple platforms (e.g., RNA-seq, protein arrays, IHC) for comprehensive analysis

• Cross-database validation: Validate findings across databases (TCGA, GTEx, in-house cohorts)

• Normal tissue controls: Always include appropriate normal tissue controls specific to each cancer type

• Technical variation: Consider platform-specific biases when comparing across different studies

• Cancer specificity: Note that EPS8L3 is upregulated in multiple cancer types including CHOL, COAD, ESCA, PAAD, and READ

How can researchers accurately quantify EPS8L3 protein levels in experimental samples?

For accurate quantification of EPS8L3 protein:

• Western blot quantification: Use densitometry software to quantify bands, normalizing to loading controls like GAPDH or β-actin

• Quantitative immunofluorescence: Employ software like ImageJ for fluorescence intensity quantification, using standardized acquisition parameters

• Flow cytometry: Quantify EPS8L3 expression levels per cell using mean fluorescence intensity

• Internal standards: Include gradient standards of known quantities to create calibration curves

• Multiple technical replicates: Perform at least three independent experiments for statistical robustness

• Standardization: Use a common positive control across all experiments for inter-experimental normalization

• Control for confounding factors: Account for cell confluence, passage number, and growth conditions that may affect expression levels

What are promising areas for future research on EPS8L3 as a therapeutic target?

Based on current knowledge, future research directions include:

• Targeted inhibition strategies: Developing specific inhibitors or antibody-drug conjugates targeting EPS8L3

• Combination therapy approaches: Investigating synergistic effects of EPS8L3 inhibition with existing HCC treatments

• Biomarker development: Validating EPS8L3 as a predictive biomarker for treatment response or recurrence

• Structure-function relationship: Elucidating the structural domains of EPS8L3 critical for its oncogenic functions

• Mechanism exploration: Further investigating how EPS8L3 affects EGFR dimerization and internalization at the molecular level

• Cancer stem cell targeting: Developing strategies to target EPS8L3 in the context of cancer stem cell populations

• Resistance mechanisms: Understanding potential resistance mechanisms to EPS8L3-targeted therapies

How can advanced microscopy techniques enhance our understanding of EPS8L3 function?

Advanced microscopy approaches for EPS8L3 research:

• Super-resolution microscopy: To visualize EPS8L3 localization relative to membrane structures and the cytoskeleton

• Live-cell imaging: To track real-time dynamics of EPS8L3 trafficking and interactions with EGFR after EGF stimulation

• FRET/FLIM analysis: To detect direct interactions between EPS8L3 and proposed binding partners

• Correlative light-electron microscopy: To understand the ultrastructural context of EPS8L3 localization

• Light sheet microscopy: For 3D visualization of EPS8L3 distribution in tumor spheroids or organoids

• Multiplexed imaging: To simultaneously visualize multiple components of the EGFR-ERK pathway in relation to EPS8L3

• Quantitative image analysis: To measure colocalization coefficients and protein proximity in various subcellular compartments

What are the best practices for validating EPS8L3 knockdown or overexpression experiments?

For properly validated genetic manipulation experiments:

• Multiple silencing strategies: Use both siRNA and shRNA approaches with at least two different sequences targeting EPS8L3

• Verification methods: Confirm knockdown or overexpression at both mRNA level (by RT-qPCR) and protein level (by Western blot)

• Specificity controls: Verify that manipulation of EPS8L3 does not affect expression of other EPS8 family members

• Rescue experiments: Perform rescue experiments by re-expressing siRNA-resistant EPS8L3 constructs

• Appropriate controls: Include proper negative controls (scrambled siRNA, empty vector)

• Time-course analysis: Monitor the duration of knockdown or overexpression effects

• Off-target effect assessment: Screen for potential off-target effects using global expression analysis

What is the optimal workflow for studying EPS8L3's role in EGFR internalization?

For studying EPS8L3's impact on EGFR internalization:

• Sample preparation: Serum-starve cells for two days before the assay to reduce basal EGFR activation

• EGF stimulation: Treat cells with purified EGF (typically 100 ng/mL) for 15 minutes

• Surface EGFR detection: Incubate non-permeabilized cells with fluorescently-labeled anti-EGFR antibodies

• Quantification method: Perform flow cytometry analysis to quantify surface EGFR levels

• Controls: Include unstimulated cells as negative controls and cells with known EGFR internalization defects as positive controls

• Complementary approaches: Confirm findings with microscopy-based internalization assays

• Time-course analysis: Analyze multiple time points after EGF stimulation to capture internalization kinetics

What experimental design best demonstrates the relationship between EPS8L3 and clinical outcomes?

To robustly demonstrate clinical relevance:

• Paired sample analysis: Use matched tumor and adjacent non-tumor tissues from the same patients

• Adequate sample size: Include sufficiently large patient cohorts with statistical power calculations

• Comprehensive clinical data: Collect complete clinicopathological information including tumor stage, grade, and survival outcomes

• Multiple cohorts: Validate findings across independent patient cohorts

• Multivariate analysis: Adjust for known prognostic factors in survival analyses

• Functional validation: Connect clinical observations with mechanistic laboratory findings

• Prospective validation: Ideally, validate retrospective findings in prospective studies