TLE4 Antibody, HRP conjugated

What is TLE4 and what cellular functions does it perform?

TLE4 (TLE Family Member 4, Transcriptional Corepressor), also known as Groucho-related protein 4, is a protein that plays a crucial role in the Notch signaling pathway. This pathway regulates cellular interactions that determine cell fate during development in both invertebrates and vertebrates . TLE4 primarily functions as a transcriptional repressor by interacting with chromatin and specifically with Histone H3, thereby influencing gene expression patterns critical for neurogenesis and epithelial differentiation .

TLE4 is predominantly expressed in the brain, particularly in the caudate nucleus, and its expression levels correlate with the differentiation status of epithelial cells . Recent research has revealed that TLE4, along with TLE3, participates in diminishing canonical Wnt signaling activity, supporting transcription of synaptic genes and acetylcholine receptor (CHRN) clustering at the neuromuscular junction .

How do HRP-conjugated TLE4 antibodies function in detection systems?

HRP (Horseradish Peroxidase) conjugated TLE4 antibodies enhance detection sensitivity through an enzymatic amplification process. In detection systems such as Cell-Based ELISA, the TLE4 protein is first captured by anti-TLE4 antibodies, which are then detected by HRP-conjugated secondary antibodies . The HRP enzyme catalyzes a colorimetric reaction upon substrate addition, generating a measurable signal that correlates with TLE4 expression levels .

This conjugation provides several advantages over unconjugated antibodies:

What are the typical applications for TLE4 antibodies in research?

TLE4 antibodies are utilized across multiple experimental platforms in both basic and translational research. According to the search results, commercially available TLE4 antibodies are validated for:

• Western blotting (WB): For protein quantification and molecular weight determination

• Immunoprecipitation (IP): For protein-protein interaction studies

• Immunofluorescence (IF): For subcellular localization studies

• Immunohistochemistry (IHC): For tissue-specific expression analysis

• Enzyme-linked immunosorbent assay (ELISA): For quantitative protein detection

Research applications span developmental biology, neuroscience, cancer research, and B-cell development studies. For instance, TLE4 antibodies have been critical in demonstrating the B-cell-restricted expression of TLE4 protein in hematopoietic cells, showing exclusive nuclear staining consistent with its co-repressive function .

How should validation of TLE4 antibody specificity be approached across different tissue types?

Validating TLE4 antibody specificity across different tissue types requires a systematic multi-method approach:

• Knockout/Knockdown Validation: The gold standard approach is validating with CRISPR/Cas9 gene-edited samples. According to source , TLE3 and TLE4 knockouts were generated using CRISPR/Cas9 gene editing in primary skeletal muscle stem cells, and knockout clones were confirmed by "absence of protein of interest by immunofluorescence microscopy and western blot" . This approach provides definitive evidence of antibody specificity.

• Transfected Lysate Testing: Compare TLE4 antibody reactivity between TLE4-transfected and non-transfected lysates. For example, source describes validation showing TLE4 antibody detection of "TLE4 expression in transfected 293T cell line... Lane 1: TLE4 transfected lysate (85.03 KDa). Lane 2: Non-transfected lysate" .

• Cross-Reactivity Assessment: Test reactivity across species and tissues where TLE4 expression is expected versus tissues where it should be absent. Source describes: "no nuclear signal was detectable on nuclei preparations from myeloblast and lymphocyte T-cell lines, confirming the B-restricted expression of the TLE4 protein in haematopoietic cells" .

• Molecular Weight Confirmation: Verify that detected bands appear at the expected molecular weight (approximately 83.8 kDa for human TLE4) .

What are the optimal normalization strategies when using TLE4 HRP-conjugated antibodies in cell-based assays?

For cell-based assays using TLE4 HRP-conjugated antibodies, multiple normalization strategies are recommended to ensure reliable results:

• GAPDH Internal Control: According to source , "Anti-GAPDH Antibody is included to serve as an internal positive control in normalizing the target absorbance values." This housekeeping protein provides a reference for relative quantification of TLE4 expression .

• Whole-Cell Normalization: "Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method is used to determine cell density. After staining, the results can be analyzed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted" .

• Multiple Loading Controls: When performing Western blots, utilize more than one loading control (e.g., β-actin in addition to GAPDH) to strengthen normalization reliability, especially when studying diverse tissue types.

• Transfected vs. Non-Transfected Controls: Include both TLE4-transfected lysates as positive controls and non-transfected lysates as negative controls, as demonstrated in validation studies: "Lane 1: TLE4 transfected lysate (85.03 KDa). Lane 2: Non-transfected lysate" .

How does TLE4 function differ from other TLE family members in developmental biology research?

TLE4 exhibits both overlapping and unique functions compared to other TLE family members in developmental processes:

The dynamic regulation of these family members highlights their importance in cellular differentiation, with TLE1 being upregulated in early differentiation stages while TLE2 and TLE4 are upregulated later, indicating roles in the transition of cells toward a terminally differentiated state .

Functionally, research has shown that "TLE3 and TLE4 participate in diminishing canonical Wnt signaling activity, supporting transcription of synaptic genes and CHRN clustering at the neuromuscular junction" . This suggests a cooperative role between these family members in specific developmental contexts.

What is the optimal protocol for using TLE4 HRP-conjugated antibodies in Western blot analysis?

For optimal Western blot results with TLE4 HRP-conjugated antibodies, the following protocol is recommended based on validated methodologies:

• Sample Preparation:

  • Prepare cell or tissue lysates using a buffer containing protease inhibitors

  • For brain tissue samples (where TLE4 is highly expressed), use gentle lysis conditions to preserve protein integrity

• Gel Electrophoresis:

  • Use 8-10% SDS-PAGE gels (TLE4 is approximately 83.8 kDa)

  • Load 20-50 μg of protein per lane

• Transfer and Blocking:

  • Transfer to PVDF membrane (recommended over nitrocellulose for this application)

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary Antibody Incubation:

  • Dilute TLE4 antibody at 1:500 as recommended in product specifications

  • Incubate overnight at 4°C with gentle agitation

• Detection:

  • For directly HRP-conjugated TLE4 antibodies, proceed directly to development

  • For two-step detection, use HRP-conjugated secondary antibody at 1:5000 dilution

  • Develop using enhanced chemiluminescence (ECL) substrate

• Controls:

  • Include TLE4-transfected lysate as positive control (shows band at 85.03 kDa)

  • Include non-transfected lysate as negative control

What are effective troubleshooting strategies for non-specific binding when using TLE4 HRP-conjugated antibodies?

When encountering non-specific binding with TLE4 HRP-conjugated antibodies, consider these evidence-based troubleshooting approaches:

• Increase Blocking Stringency:

  • Extend blocking time to 2 hours

  • Use alternative blocking agents (e.g., BSA instead of milk, or vice versa)

  • Add 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Optimize Antibody Concentration:

  • Perform titration experiments to determine optimal concentration

  • Start with recommended 1:500 dilution and adjust as needed

• Include Competitive Peptides:

  • Pre-incubate antibody with excess immunizing peptide to confirm specificity

  • Non-specific binding will persist while specific binding will be blocked

• Modify Wash Conditions:

  • Increase number of washes (5-6 times)

  • Extend wash duration (10-15 minutes each)

  • Use higher salt concentration in wash buffer (up to 500 mM NaCl) to reduce ionic interactions

• Sample Preparation Adjustments:

  • Ensure complete denaturation of proteins

  • Include additional purification steps if working with complex samples

  • Add protease inhibitors to prevent degradation products that may cross-react

• Antibody Validation:

  • Confirm antibody specificity through knockout validation methods as described in research where "Clones of each gene knockout were sequenced to confirm and identify the genomic bi-allelic mutations"

How should TLE4 antibodies be used for studying B-cell development and differentiation?

For studying B-cell development and differentiation with TLE4 antibodies, the following methodology has been validated in research:

How can TLE4 antibodies be utilized to investigate interactions between Notch and Wnt signaling pathways?

TLE4 antibodies provide valuable tools for investigating the crosstalk between Notch and Wnt signaling pathways through the following methodological approaches:

• Co-immunoprecipitation (Co-IP) Studies:

  • Use TLE4 antibodies for immunoprecipitation to pull down TLE4 protein complexes

  • Analyze co-precipitated proteins to identify interactions with components of both Notch and Wnt pathways

  • Particularly examine interactions with "CTNNB1 and TCF family members in Wnt signaling"

• Chromatin Immunoprecipitation (ChIP) Analysis:

  • Employ TLE4 antibodies to identify genomic loci where TLE4 binds

  • Compare these sites with known Notch and Wnt response elements

  • This approach leverages TLE4's role in "interacting with chromatin and specifically with Histone H3"

• Pathway Inhibition Studies:

  • Combine TLE4 detection with specific pathway inhibitors

  • Assess how modulating one pathway affects TLE4 involvement in the other

  • For example, examine how "diminishing canonical Wnt signaling activity" by TLE4 affects Notch target genes

• Dual Reporter Assays:

  • Utilize reporter constructs for both Notch and Wnt pathways

  • Measure how TLE4 manipulation affects activity of both pathways

  • This builds on findings that TLE4 "inhibits the transcriptional activation mediated by PAX5, and by CTNNB1 and TCF family members in Wnt signaling"

• Tissue-Specific Analysis:

  • Compare TLE4 pathway interactions across different tissues

  • Focus particularly on neuromuscular junctions where TLE4 has been shown to support "transcription of synaptic genes and CHRN clustering"

What role does TLE4 play in neuromuscular junction development and function?

Research has identified several critical roles for TLE4 in neuromuscular junction (NMJ) development and function:

• Regulation of Synaptic Gene Expression:

  • TLE4 supports "transcription of synaptic genes and CHRN clustering at the neuromuscular junction"

  • This function appears to be coordinated with TLE3, suggesting complementary roles

• Modulation of Canonical Wnt Signaling:

  • TLE4 participates in "diminishing canonical Wnt signaling activity" at the postsynapse

  • This modulation is crucial for proper NMJ formation and maintenance

• Response to Denervation:

  • Research using "the denervation paradigm in Axin2-lacZ reporter mice" showed "a significant decrease in canonical Wnt signaling activity"

  • This suggests TLE4 may be involved in adaptive responses to neural input loss

• Interaction with Agrin Signaling:

  • TLE4 appears to be regulated in response to agrin (AGRN), as "alterations in the transcriptional profile of the myogenic lineage in response to agrin (AGRN) suggested that TLE3 and TLE4... could be important regulators of canonical Wnt signaling activity at the postsynapse"

• Acetylcholine Receptor Clustering:

  • Experiments demonstrated that knockout of TLE4 "led to decreased AGRN-dependent acetylcholine receptor (CHRN) clustering"

  • This indicates a direct role in organizing functional components of the NMJ

This research suggests that TLE4 antibodies are valuable tools for studying NMJ development, functioning, and pathologies related to synaptic transmission defects.

How can TLE4 antibodies contribute to cancer research and potential therapeutic development?

TLE4 antibodies offer multiple avenues for advancing cancer research due to the protein's role in key developmental pathways:

• Diagnostic Biomarker Development:

  • TLE4 expression patterns may serve as diagnostic or prognostic markers

  • Research can explore "cancers where TLE family members exhibit altered expression patterns"

  • TLE4 antibodies allow precise quantification of expression levels across cancer types

• Pathway Disruption Analysis:

  • Investigate how TLE4 dysregulation affects Notch and Wnt signaling in cancer

  • These pathways are frequently altered in various malignancies

  • TLE4 antibodies enable detection of pathway component interactions in tumor samples

• Leukemia Research:

  • Given that "Diseases associated with TLE4 include Leukemia" , TLE4 antibodies are particularly valuable for hematological malignancy research

  • B-cell restricted expression of TLE4 in hematopoietic cells suggests potential roles in B-cell malignancies

• Therapeutic Target Validation:

  • TLE4 antibodies can help validate TLE4 as a potential therapeutic target

  • Monitor changes in TLE4 expression and localization in response to experimental treatments

  • Develop screening assays to identify compounds that modulate TLE4 function

• Epigenetic Regulation Studies:

  • Explore TLE4's interaction with chromatin and Histone H3 in cancer contexts

  • Investigate whether altered epigenetic regulation through TLE4 contributes to oncogenesis

  • Use ChIP assays with TLE4 antibodies to map cancer-specific binding patterns

What methodological approaches can optimize TLE4 detection in brain tissue samples?

Brain tissue presents unique challenges for protein detection due to its complexity and lipid content. For optimal TLE4 detection in brain samples:

• Tissue Preparation Optimization:

  • Use perfusion fixation when possible for animal models

  • For human samples, minimize post-mortem interval

  • Focus on caudate nucleus samples where TLE4 is "predominantly expressed"

• Antigen Retrieval Enhancement:

  • For fixed tissues, extend antigen retrieval time (20-30 minutes)

  • Test both heat-induced (citrate buffer, pH 6.0) and enzymatic (proteinase K) retrieval methods

  • The efficacy of proteinase K treatment has been demonstrated in related protocols where "diaphragms were... treated for 15 min with Proteinase K (20 μg/mL)"

• Signal Amplification Strategies:

  • Utilize tyramide signal amplification with HRP-conjugated antibodies

  • Consider biotinylated secondary antibodies with streptavidin-HRP complexes

  • These approaches enhance sensitivity while maintaining specificity

• Background Reduction Techniques:

  • Pre-block with sera from the species of secondary antibody origin

  • Include detergents like Triton X-100 at 0.1-0.3% to reduce non-specific binding

  • Use Sudan Black B (0.1-0.3%) to reduce lipofuscin autofluorescence in older brain tissues

• Multi-labeling Approaches:

  • Combine TLE4 detection with neuronal, glial, or synaptic markers

  • This contextualizes TLE4 expression within specific neural circuits

  • Particularly valuable given TLE4's role in "neurogenesis and epithelial differentiation"

Implementing these methodological refinements will maximize the sensitivity and specificity of TLE4 detection in complex brain tissue samples, enabling more accurate characterization of its expression patterns and functional roles in neurodevelopment and pathology.

What criteria should be used to evaluate the quality and specificity of commercially available TLE4 antibodies?

Researchers should assess TLE4 antibodies using these evidence-based quality control criteria:

• Validation Methods Documentation:

  • Verify antibodies have been tested in multiple applications (WB, IF, IHC, ELISA)

  • Confirm validation across species of interest (human, mouse, rat)

  • Check for knockout/knockdown validation data as the gold standard

• Specificity Assessment:

  • Review Western blot data showing a single band at the expected molecular weight (~83.8 kDa)

  • Evaluate testing in transfected versus non-transfected systems

  • Check cross-reactivity with other TLE family members (TLE1-3)

• Application-specific Performance:

  • For IHC applications, look for demonstration of expected nuclear localization pattern

  • For IF, verify proper subcellular localization showing "a dense punctate pattern to the nuclei"

  • For WB, confirm detection of the target protein at the correct molecular weight

• Technical Specifications:

  • Review antibody format (monoclonal vs. polyclonal)

  • Check host species and isotype information

  • Verify concentration and storage conditions

How does sample preparation influence the detection of TLE4 in different experimental contexts?

Sample preparation significantly impacts TLE4 detection across various experimental platforms:

• Protein Extraction Methods:

  • Nuclear extraction techniques are critical since TLE4 is a nuclear protein with "exclusive nucleus staining"

  • Standard whole-cell lysis may dilute nuclear proteins, reducing detection sensitivity

  • For brain tissue, specialized extraction buffers may be needed to overcome lipid interference

• Fixation Impact on Epitope Accessibility:

  • Paraformaldehyde fixation (4%) preserves structure but may mask epitopes

  • Research protocols show refixation in "0.2% glutaraldehyde in 4% PFA" for certain applications

  • Cold methanol fixation often provides better antibody accessibility for nuclear proteins

• Tissue-Specific Considerations:

  • B-cell samples require specialized nuclear preparation techniques to effectively detect TLE4

  • Brain tissue benefits from perfusion fixation when possible

  • Cell lines may require optimization based on expression levels

• Antigen Retrieval Requirements:

  • Heat-induced epitope retrieval may be necessary for formalin-fixed samples

  • Some protocols utilize enzymatic treatment: "treated for 15 min with Proteinase K (20 μg/mL)"

  • Optimization is needed for each tissue type and fixation method

• Storage Conditions:

  • Flash-frozen samples maintain better epitope integrity than fixed samples

  • For antibodies, following vendor recommendations to "Store at -20°C. Avoid freeze/thaw cycles" ensures optimal performance

These preparation factors must be carefully controlled and reported in research protocols to ensure reproducibility and reliable detection of TLE4 across experimental systems.