EREG Antibody, FITC conjugated

What is EREG and why is it a target for antibody-based research?

EREG (Epiregulin) is a member of the epidermal growth factor (EGF) family of cytokines that signals through the EGFR receptor. It shares significant homology with amphiregulin and plays critical roles in various physiological and pathological processes. EREG has been implicated in hepatic stellate cell (HSC) activation and liver fibrosis, as demonstrated by its significantly upregulated expression in activated HSCs compared to quiescent HSCs . Additionally, EREG is highly expressed in colorectal cancer (CRC) with minimal expression in normal tissues, making it an attractive target for cancer research and therapeutic development . Antibodies targeting EREG enable researchers to detect, quantify, and manipulate this protein in experimental systems to better understand its biological functions and pathological implications.

What are the primary research applications for EREG antibody, FITC conjugated?

EREG antibody with FITC conjugation is primarily utilized in fluorescence-based detection methods. While the search results don't explicitly list applications for the FITC-conjugated version, the non-conjugated EREG antibody is used for immunofluorescence (IF) at dilutions of 1:100-1:500 . The FITC conjugation enables direct visualization without requiring secondary antibodies, making it particularly valuable for multi-color flow cytometry, fluorescence microscopy, and high-content imaging applications. Researchers investigating EREG expression in different cell types, tissue sections, or experimental models would benefit from this ready-to-use fluorescent antibody for both qualitative and quantitative analyses.

How does FITC conjugation affect antibody performance compared to non-conjugated antibodies?

FITC (Fluorescein isothiocyanate) conjugation provides direct fluorescent detection capability but may impact antibody performance in several ways. The conjugation process can potentially affect the antibody's binding affinity or specificity if the modification occurs near the antigen-binding site. While non-conjugated antibodies (like the base EREG antibody, CSB-PA007779NA01HU) require secondary detection reagents, they often provide greater flexibility for signal amplification. Researchers should consider that FITC has an excitation maximum at approximately 495 nm and emission maximum at about 519 nm, which may present challenges in tissues with high autofluorescence. Additionally, FITC is susceptible to photobleaching, necessitating appropriate controls and imaging protocols. When selecting between conjugated and non-conjugated antibodies, researchers should consider their specific experimental design, detection sensitivity requirements, and multiplexing needs.

What protocols should be followed for optimal staining with EREG antibody, FITC conjugated?

For optimal staining with EREG antibody, FITC conjugated, researchers should adapt protocols similar to those used for other EREG antibodies with appropriate modifications for direct fluorescence detection. Based on the search results for EREG antibody applications, the following protocol elements should be considered:

For adherent cells:

• Seed cells onto poly-D-lysine-coated plates and incubate overnight to achieve appropriate confluence

• Fix cells with 4% formalin (paraformaldehyde) for 20 minutes at room temperature

• Block with 2% BSA in PBS for 5-10 minutes to reduce non-specific binding

• Incubate with EREG antibody, FITC conjugated at an appropriate dilution (starting with 1:100-1:500 as recommended for IF applications with the non-conjugated antibody) for 1-2 hours at 4°C

• Wash thoroughly with PBS to remove unbound antibody

• Mount with anti-fade mounting medium containing DAPI for nuclear counterstaining

• Image using appropriate filters for FITC detection

For flow cytometry applications, the protocol used for quantifying surface-expressed EREG molecules can be adapted:

• Prepare single-cell suspensions (1×10^6 cells/sample)

• Fix cells with dropwise addition of 10% formalin for 20 minutes

• Block with 2% BSA/PBS for 5 minutes

• Incubate with EREG antibody, FITC conjugated (at an optimized concentration) for 60 minutes at 4°C

• Wash and analyze by flow cytometry using appropriate instrument settings for FITC detection

How should researchers validate the specificity of EREG antibody, FITC conjugated?

To validate the specificity of EREG antibody, FITC conjugated, researchers should implement multiple control strategies:

• Positive and negative cell lines: Use cell lines with known EREG expression levels. The search results mention 293T cells that do not express endogenous EREG and established lines overexpressing myc-tagged hEREG or mEREG as useful systems for validation . Additionally, colorectal cancer cell lines like LoVo and DLD-1 are mentioned as EREG-expressing models .

• Competitive binding assays: Pre-incubate the antibody with recombinant EREG protein before staining to confirm that specific binding is blocked.

• EREG knockdown/knockout controls: Compare staining in wild-type cells versus cells with EREG silenced or knocked out. The search results mention that "silencing EREG significantly reduces Fe2+ staining in CAL27 and CAL33 cells" , indicating that EREG knockdown models exist and can serve as controls.

• Cross-reactivity assessment: If studying human samples, test the antibody on mouse tissues or cells to assess cross-reactivity, as some EREG antibodies (like H231) bind both human and mouse EREG with high affinity .

• Secondary-only controls: For methodological validation, include samples stained with an isotype-matched FITC-conjugated control antibody to assess non-specific binding.

What quantification methods are appropriate for EREG expression analysis using FITC-conjugated antibodies?

For quantitative analysis of EREG expression using FITC-conjugated antibodies, researchers can employ several approaches depending on their application:

• Flow cytometry quantification: The search results describe a method for quantifying surface-expressed EREG molecules per cell using PE-conjugated antibodies and QuantiBRITE PE beads to generate a standard curve . This approach can be adapted for FITC-conjugated antibodies using appropriate FITC calibration beads. The general workflow involves:

  • Running quantification beads with known FITC molecules per bead

  • Generating a standard curve plotting Log10 of FITC molecules/bead versus Log10 of geometric means

  • Using the equation y=mx+c to calculate the number of bound antibodies per cell

• Fluorescence microscopy quantification:

  • Capture images using standardized exposure settings

  • Use image analysis software to measure mean fluorescence intensity within defined regions of interest

  • Include appropriate background subtraction methods

  • Normalize to cell number or area as needed

• Plate reader-based quantification: For high-throughput analysis, researchers can adapt the method described where "fluorescence intensity was quantified using Tecan Infinite M1000 plate reader" following antibody staining of cells in 96-well plates.

How can EREG antibody, FITC conjugated be employed in studying liver fibrosis mechanisms?

EREG antibody, FITC conjugated can be a valuable tool for investigating liver fibrosis mechanisms, particularly focusing on hepatic stellate cell (HSC) activation. Based on the search results, EREG expression is significantly upregulated during HSC activation both in vivo and in vitro, and this process is regulated by myocardin-related transcription factor A (MRTF-A) .

Researchers can employ FITC-conjugated EREG antibodies in the following approaches:

• Temporal expression analysis: Track EREG expression changes during HSC activation by isolating primary HSCs from normal and fibrotic livers (e.g., from CCl4-treated mice) at different time points and analyzing EREG expression using flow cytometry or immunofluorescence microscopy .

• Co-localization studies: Combine EREG antibody, FITC conjugated with antibodies against HSC activation markers (e.g., α-SMA/ACTA2) labeled with spectrally distinct fluorophores to examine the correlation between EREG expression and HSC activation status.

• Mechanistic investigations: Explore the EREG-MRTF-A feedforward loop described in the research. Researchers could use the FITC-conjugated EREG antibody to monitor changes in EREG expression following manipulations of MRTF-A (e.g., MRTF-A depletion or inhibition) or following EREG treatment which stimulates nuclear translocation of MRTF-A .

• Therapeutic intervention assessment: Evaluate the efficacy of potential anti-fibrotic compounds by monitoring changes in EREG expression as an indicator of HSC activation status. The search results suggest that "targeting the EREG-MRTF-A axis may yield therapeutic solutions against liver fibrosis" .

What approaches can be used to study EREG antibody internalization dynamics in cancer cells?

Understanding antibody internalization dynamics is critical for developing effective antibody-drug conjugates (ADCs) and therapeutic strategies. Based on the search results, the EREG monoclonal antibody H231 "internalized to lysosomes, which is important for ADC payload release" . Researchers can adapt these findings to study internalization dynamics using FITC-conjugated EREG antibodies:

• Time-course internalization assays:

  • Incubate live cells with EREG antibody, FITC conjugated at 4°C to allow surface binding without internalization

  • Shift cells to 37°C to permit internalization and collect samples at different time points

  • Use confocal microscopy to visualize changes in antibody localization from membrane to intracellular compartments

• Co-localization with endocytic markers:

  • Perform dual staining with EREG antibody, FITC conjugated and markers for different endocytic compartments (early endosomes, late endosomes, lysosomes)

  • Use spectral unmixing if needed to distinguish FITC signal from other fluorophores

  • Quantify co-localization coefficients over time to track the trafficking pathway

• Acid wash technique to distinguish surface vs. internalized antibody:

  • After incubation with EREG antibody, FITC conjugated, treat one set of samples with acid buffer (pH 2.5-3.0) to strip surface-bound antibody

  • Compare with untreated samples to quantify the internalized fraction

  • Track changes in the internalization rate over time

• pH-sensitive fluorescent probes:

  • Use dual-labeled antibodies (FITC plus a pH-insensitive dye) to distinguish between surface-bound and internalized antibodies based on the quenching of FITC fluorescence in acidic endosomal/lysosomal compartments

How can EREG antibody, FITC conjugated contribute to colorectal cancer research?

EREG antibody, FITC conjugated can significantly contribute to colorectal cancer (CRC) research based on the findings that "EREG is highly expressed in RAS wildtype and mutant CRC with minimal expression in normal tissues" . This makes it both an interesting biomarker and therapeutic target. Researchers can utilize FITC-conjugated EREG antibodies in the following applications:

• Expression profiling across CRC subtypes:

  • Analyze EREG expression levels in different CRC cell lines and patient samples using flow cytometry

  • Correlate expression levels with RAS mutational status, as the search results indicate EREG is expressed in both RAS wildtype and mutant CRC

  • Quantify the number of EREG molecules per cell using calibration methods described in the search results

• Patient stratification biomarker development:

  • Develop standardized protocols for EREG detection in patient samples

  • Establish expression thresholds that might predict responsiveness to EREG-targeted therapies

  • Compare EREG expression with other EGFR pathway components

• Therapeutic response monitoring:

  • Assess changes in EREG expression following treatment with various anticancer agents

  • Monitor the effects of EREG-targeted antibody-drug conjugates on EGFR pathway activity

  • As noted in the search results, EREG ADCs "neutralized EGFR pathway activity" and showed efficacy "irrespective of RAS mutations"

• Tumor microenvironment studies:

  • Investigate the relationship between EREG expression and tumor microenvironment factors

  • Combined with other markers, analyze how EREG expression correlates with immune cell infiltration or exclusion

What are common technical challenges when using EREG antibody, FITC conjugated, and how can they be addressed?

Researchers working with EREG antibody, FITC conjugated may encounter several technical challenges:

• Signal sensitivity issues:

  • Problem: FITC has moderate brightness and is susceptible to photobleaching

  • Solution: Use anti-fade mounting media, optimize acquisition settings, consider alternative conjugates for very low expression targets

• Background fluorescence:

  • Problem: Tissue autofluorescence in the FITC channel, particularly in liver tissue which is relevant for EREG-related liver fibrosis studies

  • Solution: Include proper autofluorescence controls, consider spectral unmixing approaches, or use Sudan Black B to reduce autofluorescence

• Fixation artifacts:

  • Problem: Over-fixation can mask epitopes and reduce antibody binding

  • Solution: Optimize fixation conditions; the search results indicate successful staining with 10-20 minutes of formalin fixation

• Species cross-reactivity:

  • Problem: Uncertainty about antibody specificity between human and mouse EREG

  • Solution: Validate cross-reactivity experimentally; the search results mention that some EREG antibodies like H231 bind both human EREG (hEREG) and mouse EREG (mEREG) with high affinity

• Quantification accuracy:

  • Problem: Difficulty in standardizing measurements across experiments

  • Solution: Implement calibration standards as described in the QuantiBRITE approach mentioned in the search results

How should discrepancies between EREG protein detection methods be reconciled?

When faced with discrepancies between different EREG detection methods, researchers should systematically investigate the source of variation:

• Validate antibody performance across methods:

  • Compare EREG antibody, FITC conjugated results with other detection methods such as ELISA, which was used to confirm EREG protein levels in activated HSCs in the search results

  • For each method, include positive and negative controls with known EREG expression levels

• Consider epitope accessibility differences:

  • Different detection methods may access different epitopes

  • The conformation of EREG may vary between methods (native vs. denatured)

  • Examine whether the antibody recognizes both pro-EREG and mature, cleaved EREG as mentioned in the search results

• Evaluate temporal dynamics:

  • EREG expression changes during processes like HSC activation

  • Ensure samples are collected at comparable time points when comparing methods

• Implement orthogonal validation:

  • Confirm results using gene expression analysis (e.g., RT-PCR)

  • Use genetic approaches such as the EREG silencing mentioned in the reviews

• Document methodological details:

  • Record precise fixation conditions, antibody concentrations, and incubation times

  • The search results describe specific protocols (e.g., 4% formalin fixation, 2% BSA blocking, 1-2 hour antibody incubation at 4°C) that should be consistently followed

What statistical approaches are appropriate for analyzing EREG expression data from imaging or flow cytometry?

For robust statistical analysis of EREG expression data using FITC-conjugated antibodies, researchers should consider:

• Flow cytometry data analysis:

  • Report both percentage of positive cells and median fluorescence intensity (MFI)

  • Subtract isotype control or unstained control MFI values to obtain specific signal

  • For comparing multiple groups, use appropriate statistical tests (ANOVA with post-hoc tests for multiple comparisons)

  • Consider displaying data as both histograms and quantitative bar graphs with error bars

• Imaging data analysis:

  • Establish consistent thresholding criteria for identifying positive signals

  • Quantify parameters such as intensity, area, and co-localization with other markers

  • Analyze sufficient numbers of cells or fields to achieve statistical power

  • Consider hierarchical analysis if working with tissues (e.g., nested design accounting for multiple measurements within the same sample)

• Correlation analyses:

  • When investigating relationships between EREG expression and other parameters (e.g., MRTF-A levels ), use appropriate correlation statistics (Pearson's or Spearman's depending on data distribution)

  • Present data in scatter plots with regression lines and confidence intervals

• Longitudinal studies:

  • For experiments tracking EREG expression over time (e.g., during HSC activation ), consider repeated measures ANOVA or mixed models

  • Account for potential batch effects if samples are processed on different days

• Sample size considerations:

  • Perform power analysis to determine appropriate sample sizes

  • Report both biological and technical replicates clearly

How is EREG antibody being used in the development of antibody-drug conjugates for cancer therapy?

The search results provide substantial information about using EREG antibodies to develop antibody-drug conjugates (ADCs) for cancer therapy, particularly colorectal cancer. While this research used non-FITC conjugated antibodies, the findings provide important context for researchers using FITC-conjugated antibodies in related studies:

• Target validation and biodistribution:

  • EREG antibody H231 showed "high specificity and affinity for human and mouse EREG"

  • ImmunoPET and ex vivo biodistribution studies demonstrated "significant tumor uptake of 89Zr-labeled H231 with minimal uptake in normal tissues"

  • Researchers can use FITC-conjugated EREG antibodies in preclinical studies to visualize biodistribution and tumor uptake using fluorescence imaging

• Internalization dynamics:

  • H231 "internalized to lysosomes, which is important for ADC payload release"

  • FITC-conjugated EREG antibodies can be used to study internalization kinetics and intracellular trafficking through live-cell imaging

• Linker-payload optimization:

  • The search results describe development of EREG ADCs with various linkers (dipeptide and tripeptide) and payloads (duocarmycin DM)

  • Researchers can use FITC-conjugated EREG antibodies to study how different chemical modifications affect antibody binding and internalization properties

• Therapeutic efficacy:

  • EREG ADCs demonstrated "potent and selective growth inhibition in cancer cell lines and patient-derived tumor xenograft (PDX) models of CRC with or without mutant RAS"

  • FITC-conjugated EREG antibodies can help monitor target engagement and expression levels before and after treatment

• Safety assessment:

  • The search results indicate EREG ADCs were "well-tolerated" in preclinical models

  • Researchers can use FITC-conjugated EREG antibodies to monitor potential off-target binding in normal tissues

What role might EREG play in the tumor microenvironment and immune response?

While the search results don't directly address EREG's role in the tumor microenvironment and immune response, researchers can investigate these aspects using FITC-conjugated EREG antibodies:

• EREG expression in stromal and immune cells:

  • Use FITC-conjugated EREG antibodies in combination with markers for different cell types in the tumor microenvironment

  • Investigate whether tumor-associated macrophages, cancer-associated fibroblasts, or other stromal cells express EREG

  • The search results show EREG plays a role in hepatic stellate cell activation , suggesting it may have functions in other stromal cells

• EREG and immune checkpoint regulation:

  • Explore potential relationships between EREG expression and immune checkpoint molecules

  • Investigate whether EREG expression correlates with immune cell infiltration or exclusion in colorectal cancer samples

  • Study how EREG-targeted therapies might affect the immune microenvironment

• EREG in therapy resistance mechanisms:

  • Investigate whether EREG expression changes following immunotherapy or other cancer treatments

  • Explore potential roles of EREG in developing resistance to EGFR-targeted therapies

  • The search results indicate that "EREG ADCs may show promise for both RAS mutant and wildtype patients, thus improving existing treatment options"

• EREG and inflammation:

  • Study EREG's role in inflammatory processes within the tumor microenvironment

  • Investigate relationships between inflammatory stimuli and EREG expression

  • The search results link EREG to liver fibrosis , which often involves inflammatory processes

What technical advances might improve EREG detection and quantification in the future?

Several technical advances could enhance EREG detection and quantification beyond current capabilities of FITC-conjugated antibodies:

• Advanced fluorophore conjugations:

  • Development of EREG antibodies conjugated to brighter, more photostable fluorophores than FITC

  • Expansion to near-infrared fluorophores for improved tissue penetration in imaging applications

  • Creation of photoactivatable or photoswitchable EREG antibody conjugates for super-resolution microscopy

• Multiplexed detection systems:

  • Development of multiplexed panels including EREG and other EGFR pathway components

  • Integration with mass cytometry (CyTOF) or imaging mass cytometry for highly multiplexed analysis

  • Spatial transcriptomics approaches that combine EREG protein detection with EREG mRNA visualization

• Advanced imaging technologies:

  • Adaptation of EREG antibodies for expansion microscopy to visualize subcellular localization

  • Development of EREG-targeting nanobodies or small recombinant antibody fragments for improved tissue penetration

  • Integration with intravital microscopy to monitor EREG dynamics in living organisms

• Computational analysis improvements:

  • Machine learning algorithms to automatically identify and quantify EREG-positive cells in complex tissues

  • Systems biology approaches to integrate EREG expression data with other -omics datasets

  • Digital pathology platforms for standardized EREG quantification across laboratories

• Novel functional assays:

  • Development of biosensors to monitor EREG-EGFR signaling dynamics in real-time

  • CRISPR-based reporters for visualizing endogenous EREG expression

  • Proximity ligation assays to detect EREG interactions with binding partners like MRTF-A mentioned in the search results

What are the key methodological considerations for researchers new to working with EREG antibodies?

Researchers new to working with EREG antibodies should consider the following methodological recommendations:

• Proper validation is essential: Before proceeding with experiments, validate the specificity of your EREG antibody using positive and negative controls. The search results mention 293T cells that don't express endogenous EREG as a potential negative control, and 293T cells overexpressing myc-tagged hEREG or mEREG as positive controls .

• Consider species cross-reactivity: Verify whether your EREG antibody recognizes human EREG, mouse EREG, or both. The search results indicate that antibodies like H231 bind both human and mouse EREG with high affinity , which can be advantageous for translational research.

• Optimize fixation and staining conditions: The search results describe successful protocols using 4-10% formalin fixation for 20 minutes, followed by blocking with 2% BSA/PBS, and antibody incubation at 4°C . Start with recommended dilutions (1:100-1:500 for immunofluorescence applications) and optimize for your specific experimental system.

• Quantify expression levels appropriately: Consider using standardized methods to quantify EREG expression, such as the QuantiBRITE approach described in the search results for determining molecules per cell .

• Include appropriate controls: Use isotype controls, secondary-only controls (for non-conjugated antibodies), and biological controls (EREG knockdown or knockout) to ensure the specificity of your results.

What are the most promising future research directions for EREG antibody applications?

Based on the search results and current research trends, the following represent promising future directions for EREG antibody applications:

• Therapeutic development: The search results demonstrate that "EREG-targeting antibody-drug conjugates demonstrate acceptable safety and robust therapeutic efficacy in RAS mutant and wildtype colorectal cancer" . This suggests significant potential for developing EREG-targeted therapies for cancers expressing high levels of EREG.

• Biomarker development: EREG expression levels could serve as biomarkers for disease progression or treatment response, particularly in liver fibrosis and colorectal cancer . FITC-conjugated EREG antibodies could facilitate high-throughput screening of patient samples.

• Mechanistic studies: Further investigation of the "EREG-MRTF-A feedforward loop that contributes to HSC activation" could provide insights into fibrotic diseases. Similarly, studying EREG's role in EGFR signaling in various cancers could identify new therapeutic targets.

• Combination therapy approaches: Exploring how EREG-targeted therapies might synergize with other treatment modalities, such as immunotherapy or chemotherapy, represents an important research direction.

• Technical innovations: Development of new EREG detection methods with improved sensitivity, specificity, and multiplexing capabilities could advance our understanding of EREG biology in complex tissues and disease states.

How can researchers integrate EREG studies with broader investigations of EGFR signaling pathways?

Researchers can integrate EREG studies with broader EGFR signaling pathway investigations through several approaches:

• Comparative analysis with other EGFR ligands: Study EREG alongside other EGFR ligands such as amphiregulin, which the search results note "plays critical roles in HSC activation and liver fibrosis" . Compare their expression patterns, binding affinities, and downstream signaling effects.

• Pathway-wide phosphorylation analysis: Combine EREG detection with phospho-specific antibodies targeting different components of the EGFR signaling cascade to understand how EREG influences pathway activation.

• Genetic manipulation studies: Use CRISPR-based approaches to modify EREG, EGFR, or downstream effectors and study the consequences on cellular phenotypes relevant to diseases like liver fibrosis or colorectal cancer .

• Systems biology approaches: Integrate EREG expression data with transcriptomic, proteomic, and phosphoproteomic datasets to build comprehensive models of EGFR signaling networks in different biological contexts.

• Translational research applications: Connect basic research findings to clinical applications, such as developing combination therapies that target both EREG and other components of the EGFR pathway. The search results indicate that "EREG ADCs may show promise for both RAS mutant and wildtype patients, thus improving existing treatment options" compared to traditional EGFR-targeted therapies.