ZEB1 Antibody, HRP conjugated

What is ZEB1 and what biological functions does it regulate?

ZEB1 (also known as AREB6, BZP, DELTAEF1, FECD6, NIL2A, PPCD3, TCF8, ZFHEP, and ZFHX1A) is a pivotal non-receptor transcription factor that regulates gene expression during embryonic development, particularly within mesodermal tissues, and in the maturation of various tissues. Structurally, ZEB1 contains two zinc finger domains essential for DNA binding and transcriptional repression, a homeodomain involved in protein-protein interactions, and three repression domains that facilitate modulating gene expression through mechanisms such as site competition and enhancer silencing . ZEB1 functions primarily as a transcriptional repressor, inhibiting interleukin-2 gene expression and repressing E-cadherin promoter activity, which induces epithelial-mesenchymal transition (EMT) . In neuronal development, ZEB1 controls differentiation by transcriptionally repressing polarity genes in neuronal progenitors, inhibiting polarization and retaining progenitors in their germinal zone .

What applications is ZEB1 Antibody, HRP conjugated suitable for?

ZEB1 Antibody, HRP conjugated combines the specificity of anti-ZEB1 antibody with the signal amplification capabilities of horseradish peroxidase enzyme, making it suitable for multiple applications, including:

• Western blotting (WB): Direct detection without secondary antibody requirements

• Immunohistochemistry (IHC): Enhanced sensitivity for tissue section analysis

• Enzyme-linked immunosorbent assay (ELISA): Direct detection with chromogenic substrates

• Immunocytochemistry (ICC): Cellular localization studies with chromogenic or chemiluminescent detection

The antibody is designed to detect human ZEB1 with exceptional sensitivity and reliability across these applications . When using this conjugate, researchers can simplify their detection protocols by eliminating secondary antibody incubation steps while maintaining high specificity for their target protein.

What controls should be included when using ZEB1 Antibody, HRP conjugated?

When designing experiments with ZEB1 Antibody, HRP conjugated, the following controls should be implemented:

Positive Controls:

• Cell lines with known ZEB1 expression (e.g., mesenchymal cancer cell lines)

• Tissues with documented ZEB1 expression (e.g., developing mesoderm)

• Recombinant ZEB1 protein standards for quantification

Negative Controls:

• Isotype-matched HRP-conjugated control antibody

• Samples pre-treated with ZEB1 blocking peptide

• Cell lines with ZEB1 knockdown via siRNA or CRISPR

• Epithelial cell lines with minimal ZEB1 expression

Additionally, when studying ZEB1 in the context of neuronal differentiation, cerebellum samples can serve as excellent developmental controls, as ZEB1 expression is high in granule neuron progenitors but decreases during differentiation .

What sample preparation methods optimize ZEB1 detection?

Optimal sample preparation depends on the application:

For Western Blotting:

• Use RIPA or NP-40 lysis buffers supplemented with protease inhibitors

• Include phosphatase inhibitors if studying ZEB1 phosphorylation status

• Use fresh samples when possible; ZEB1 may degrade in long-term storage

• Heat samples at 95°C for 5 minutes in reducing sample buffer

• Load 20-50 μg total protein per lane for adequate detection

For Immunohistochemistry:

• Fix tissues in 4% paraformaldehyde

• For paraffin sections, include antigen retrieval steps (citrate buffer pH 6.0)

• Block endogenous peroxidase with 3% hydrogen peroxide

• Include permeabilization steps with 0.1-0.3% Triton X-100

• Use protein blocking solutions containing 5-10% serum

Proper sample preparation ensures preservation of ZEB1 epitopes and minimizes background signal during detection procedures.

How can I optimize signal-to-noise ratio when using ZEB1 Antibody, HRP conjugated?

To achieve optimal signal-to-noise ratio:

• Optimize antibody concentration through titration experiments (typically 1:100-1:1000 dilution range)

• Extend blocking time to reduce non-specific binding (1-2 hours at room temperature)

• Include 0.05-0.1% Tween-20 in wash buffers to reduce background

• For Western blots, use 5% non-fat milk or BSA in TBS-T for blocking

• For IHC/ICC, minimize endogenous peroxidase activity with hydrogen peroxide pre-treatment

• Use fresh substrate solutions for optimal signal development

• Optimize substrate incubation time to prevent overdevelopment

For chemiluminescent detection, incorporate graduated exposure times to identify the optimal signal window before saturation occurs.

How can ZEB1 Antibody, HRP conjugated be utilized to study epithelial-mesenchymal transition in cancer models?

ZEB1 serves as a master regulator of epithelial-mesenchymal transition, contributing to tumor progression and metastasis by promoting EMT—a process that enables epithelial cells to acquire mesenchymal traits, enhancing their migratory capabilities . When investigating EMT using ZEB1 Antibody, HRP conjugated:

• Multiplex protein analysis approach:

  • Co-stain for ZEB1 along with epithelial markers (E-cadherin, cytokeratins) and mesenchymal markers (N-cadherin, vimentin)

  • Use serial sections with HRP-conjugated antibodies against each marker

  • Quantify inverse correlation between ZEB1 and epithelial markers

• Time-course analysis methodology:

  • Treat epithelial cancer cells with EMT inducers (TGF-β, hypoxia)

  • Collect samples at regular intervals (0, 12, 24, 48, 72 hours)

  • Track ZEB1 upregulation in relation to E-cadherin downregulation

  • Correlate these changes with altered cell morphology and invasion capacity

• ZEB1 target gene analysis:

  • Monitor ZEB1 binding to E-box elements in the promoters of epithelial genes

  • Use ChIP assays with anti-ZEB1 followed by qPCR for specific promoters

  • Correlate ZEB1 binding with transcriptional repression of target genes

This multi-dimensional approach leverages the specificity and convenience of HRP-conjugated ZEB1 antibody to comprehensively characterize EMT transitions in cancer models.

What methodological approaches can resolve conflicting ZEB1 expression data between different detection systems?

Researchers occasionally encounter conflicting data when comparing ZEB1 expression across different detection platforms. To resolve such discrepancies:

• Epitope mapping validation:

  • Compare the epitope recognized by your HRP-conjugated antibody (amino acids 39-140 of human ZEB1 ) with other antibodies

  • Perform epitope competition assays using recombinant ZEB1 fragments

  • Verify accessibility of the epitope under different sample preparation conditions

• Isoform-specific detection strategy:

  • ZEB1 has multiple isoforms and splice variants

  • Design PCR primers to quantify specific isoform expression at mRNA level

  • Compare protein detection patterns with predicted molecular weights of known isoforms

  • Use multiple antibodies targeting different ZEB1 domains to confirm expression patterns

• Cross-validation protocol:

  • Implement at least three independent detection methods:

    • Western blot with HRP-conjugated antibody

    • Immunofluorescence with different antibody clone

    • qRT-PCR for ZEB1 mRNA

  • Confirm specificity using ZEB1 knockdown or knockout samples

• Standardization of quantification:

  • Establish a standard curve using recombinant ZEB1 protein

  • Normalize ZEB1 signals to appropriate loading controls

  • Use digital image analysis with consistent thresholding parameters

This systematic approach helps identify the source of discrepancies and establishes reliable ZEB1 expression data across different experimental platforms.

How can ZEB1 Antibody, HRP conjugated be applied to study neurodevelopmental processes?

ZEB1 plays a critical role in neuronal differentiation by controlling polarity gene expression in neuronal progenitors. For neurodevelopmental studies:

• Cerebellar development analysis protocol:

  • Investigate ZEB1 expression in cerebellar granule neuron progenitors (GNPs)

  • Track expression during differentiation stages using timed developmental samples

  • Correlate ZEB1 levels with progenitor markers (Ki67) and differentiation markers (TAG1, NeuN)

• Ex vivo cerebellar slice methodology:

  • Prepare cerebellar slice cultures from P7 mice

  • Manipulate ZEB1 expression using electroporation techniques

  • Monitor cellular migration, differentiation, and germinal zone exit

  • Quantify metrics like migration distance (34 ± 10 μm in control vs. 68 ± 18 μm in ZEB1-silenced cells)

• Polarity complex analysis:

  • Examine correlation between ZEB1 and polarity complex proteins (PARD6A, PARD3A, DLG2, LIN7A)

  • Implement co-detection methods to visualize mutual exclusivity of expression

  • Quantify inverse correlation between ZEB1 and polarity genes during development

• Sonic Hedgehog pathway interaction assessment:

  • Study ZEB1 expression in response to SHH pathway activation

  • Use SHH agonists (e.g., SAG) to upregulate ZEB1 in GNPs

  • Measure corresponding downregulation of polarity proteins like PARD6A

These approaches utilize the specificity of HRP-conjugated ZEB1 antibody to explore the molecular mechanisms governing neuronal differentiation and germinal zone exit during brain development.

What techniques can optimize ZEB1 Antibody, HRP conjugated for chromatin immunoprecipitation studies?

While HRP-conjugated antibodies are not typically used for chromatin immunoprecipitation (ChIP), researchers interested in ZEB1 binding sites may adapt the following protocol:

• Modified ChIP workflow:

  • Use unconjugated ZEB1 primary antibody for the immunoprecipitation step

  • Follow standard ChIP protocols for crosslinking, sonication, and IP

  • Reserve HRP-conjugated ZEB1 antibody for validation experiments:

    • Western blot analysis of ChIP input and IP fractions

    • Dot blot verification of enriched chromatin fragments

• ZEB1 binding site validation strategy:

  • Focus on E-box elements (5'-CANNTG-3') in target gene promoters

  • Design primers flanking predicted ZEB1 binding sites in:

    • E-cadherin promoter

    • IL-2 gene regulatory regions

    • Polarity gene promoters (PARD6A, PARD3A, DLG2, LIN7A)

  • Use sequential ChIP to investigate co-occupancy with co-repressors like CtBP

• Data analysis approach:

  • Normalize ChIP-qPCR data to input and IgG controls

  • Compare enrichment at target sites versus non-specific genomic regions

  • Correlate binding with transcriptional repression of target genes

This integrated approach leverages both unconjugated and HRP-conjugated ZEB1 antibodies for comprehensive characterization of ZEB1's transcriptional regulatory network.

What are the considerations for using ZEB1 Antibody, HRP conjugated in medulloblastoma research?

ZEB1 expression is significantly elevated in the Sonic Hedgehog (SHH) medulloblastoma subgroup, which originates from granule neuron progenitors with persistent SHH activation . When studying medulloblastoma:

• Subgroup classification methodology:

  • Implement ZEB1 immunohistochemistry as part of a marker panel for medulloblastoma subgrouping

  • Quantify ZEB1 expression levels using digital pathology techniques

  • Correlate with other SHH pathway markers (GLI1, PTCH1)

  • Note that ZEB1 RNA is approximately four times higher in SHH medulloblastoma compared to WNT, Group3, and Group4 subtypes

• Differentiation therapy investigation approach:

  • Study ZEB1 target genes as potential differentiation therapy targets

  • Monitor polarity gene expression (PARD6A, PARD3A) in response to differentiation agents

  • Assess germinal zone exit capability after target restoration

  • Quantify differentiation markers (TUJ1) in relation to ZEB1 expression

• Medulloblastoma mouse model analysis protocol:

  • Examine ZEB1 expression in Ptch1+/-, Cdkn2c-/- SHH medulloblastoma mouse models

  • Implement tissue microarray technology for high-throughput analysis

  • Compare expression patterns with normal cerebellar development

  • Note the complementary expression pattern between ZEB1 and neuronal differentiation marker TUJ1

• Therapeutic response assessment:

  • Monitor ZEB1 expression changes in response to SHH pathway inhibitors

  • Correlate decreased ZEB1 levels with increased polarity gene expression

  • Assess differentiation status and proliferation markers

  • Evaluate tumor invasiveness in relation to ZEB1 activity

These approaches utilize ZEB1 Antibody, HRP conjugated as a valuable tool for exploring the molecular mechanisms underlying medulloblastoma development and for evaluating potential differentiation-based therapeutic strategies.

How can non-specific background be minimized when using ZEB1 Antibody, HRP conjugated?

When encountering high background with ZEB1 Antibody, HRP conjugated:

• Optimization protocol:

  • Increase blocking time and concentration (5-10% normal serum or BSA for 1-2 hours)

  • Add 0.1-0.3% Triton X-100 to permeabilize cells before antibody incubation

  • Implement more stringent washing (5-6 washes of 5-10 minutes each)

  • Pre-absorb antibody with tissue powder from ZEB1-negative samples

  • Reduce primary antibody concentration (perform titration series)

• Endogenous peroxidase management:

  • For tissues or cells with high endogenous peroxidase activity:

    • Incubate with 0.3-3% hydrogen peroxide for 10-30 minutes before blocking

    • Consider using peroxidase inhibitors like phenylhydrazine for red blood cells

    • For plant samples or chlorophyll-containing tissues, include sodium azide treatment

• Alternative detection strategies:

  • Switch to biotin-free detection systems if streptavidin-binding proteins cause background

  • Consider fluorescent secondary antibodies if autofluorescence is not a concern

  • Implement Sudan Black B treatment to reduce autofluorescence in tissues

Systematic optimization of these parameters will significantly reduce non-specific background while preserving specific ZEB1 signals.

What strategies can address weak or absent ZEB1 signal detection?

When ZEB1 signal is weak or undetectable:

• Epitope retrieval enhancement:

  • For formalin-fixed tissues, extend heat-induced epitope retrieval time

  • Test multiple retrieval buffers (citrate pH 6.0, EDTA pH 8.0, Tris-EDTA pH 9.0)

  • Implement pressure cooker or microwave-based retrieval methods

  • For frozen sections, test different fixation protocols that preserve epitope accessibility

• Signal amplification approaches:

  • Implement tyramide signal amplification (TSA) technology

  • Use polymer-HRP detection systems for enhanced sensitivity

  • Consider avidin-biotin complex (ABC) method for additional amplification

  • Extend substrate development time with careful monitoring

• Sample preparation modifications:

  • For Western blots, increase protein loading (50-100 μg per lane)

  • Reduce detergent concentration in lysis buffers

  • Avoid freeze-thaw cycles that may degrade ZEB1 protein

  • For low-abundance samples, implement immunoprecipitation before Western blotting

These approaches systematically address potential causes of weak signal and implement appropriate enhancements to achieve robust ZEB1 detection.

How can ZEB1 Antibody, HRP conjugated be utilized in multiplexed detection systems?

For comprehensive analysis of ZEB1 in relation to other markers:

• Sequential multiplexing protocol:

  • Implement tyramide signal amplification (TSA) with different fluorophores

  • Perform sequential antibody staining-stripping cycles

  • Use spectral unmixing to resolve overlapping signals

  • Quantify co-localization or mutual exclusivity patterns

  • Compare ZEB1 with EMT markers or polarity proteins in the same sample

• Chromogenic multiplexing approach:

  • Use orthogonal detection systems (HRP, alkaline phosphatase)

  • Implement distinct substrate chromogens (DAB, FastRed, Vector Blue)

  • Optimize deposition sequence from lightest to darkest chromogen

  • Counterstain with hematoxylin for nuclear visualization

  • Utilize digital separation algorithms for quantitative analysis

• Mass cytometry application:

  • Conjugate ZEB1 antibody with rare earth metals

  • Implement CyTOF technology for highly multiplexed analyses

  • Simultaneously detect up to 40 markers in single cells

  • Apply dimensionality reduction algorithms for data visualization

These multiplexing approaches enable comprehensive characterization of ZEB1's relationships with interacting proteins and signaling pathways in complex biological systems.

What is the potential for ZEB1 Antibody, HRP conjugated in liquid biopsy applications?

Emerging research suggests potential applications for ZEB1 detection in liquid biopsies:

• Circulating tumor cell (CTC) analysis methodology:

  • Capture CTCs using epithelial markers (EpCAM, cytokeratins)

  • Implement ZEB1 immunocytochemistry to identify EMT-undergoing CTCs

  • Quantify ZEB1-positive CTCs as a marker of aggressive disease

  • Correlate ZEB1 expression with treatment resistance or metastatic potential

• Exosome characterization approach:

  • Isolate tumor-derived exosomes from patient plasma

  • Analyze ZEB1 protein or mRNA content

  • Implement nanoparticle flow cytometry for exosome characterization

  • Correlate exosomal ZEB1 with disease progression

• Cell-free DNA methylation analysis:

  • Examine methylation status of ZEB1 promoter in cfDNA

  • Correlate with ZEB1 expression levels in primary tumors

  • Monitor changes during treatment as a resistance biomarker

While these applications remain largely experimental, they represent promising directions for translating ZEB1 antibody technologies into clinical applications for cancer monitoring and management.

ZEB1 Target Gene Expression Patterns in Neuronal Development

Data derived from experimental findings showing ZEB1's role in repressing polarization and differentiation genes in cerebellar granule neuron progenitors .

Effect of ZEB1 Manipulation on Neuronal Migration in Cerebellar Slice Cultures

Data demonstrates ZEB1's role in inhibiting differentiation and germinal zone exit of cerebellar granule neuron progenitors .

ZEB1 Expression in Medulloblastoma Subgroups

Data shows approximately four-fold higher ZEB1 expression in SHH medulloblastoma compared to other subgroups, supporting the link between SHH pathway activation and ZEB1 expression .