TLE4 Antibody, Biotin conjugated

What is TLE4 and what are its primary functions in cellular signaling?

TLE4 (Transducin-Like Enhancer protein 4) functions as a transcriptional corepressor that plays crucial roles in multiple signaling pathways. TLE4 is a human protein with a molecular weight of approximately 83.8 kilodaltons that belongs to the Groucho/TLE family of transcriptional regulators .

It functions primarily by:

• Binding to a variety of transcription factors, including PAX5

• Inhibiting transcriptional activation mediated by CTNNB1 and TCF family members in Wnt signaling

• Acting as an essential component for the transcriptional repressor activity of SIX3 during retina and lens development

• Participating in the Notch signaling pathway, which regulates cellular interactions determining cell fate during development

TLE4 is also known by several aliases including Groucho-related protein 4 (GRG4), transducin-like enhancer of split 4, E(spI), and BCE-1 .

What applications are most suitable for biotin-conjugated TLE4 antibodies?

Based on available research data, biotin-conjugated TLE4 antibodies are particularly useful for:

Primary Applications:

• ELISA (Enzyme-Linked Immunosorbent Assay) - The biotin conjugation enhances sensitivity in detection systems

• Immunofluorescence (IF) - Biotin-conjugated antibodies allow for signal amplification through streptavidin-based detection systems

• Multiplex immunoassays - When used alongside non-biotinylated primary antibodies for other targets

Tissue-specific Applications:

• B-cell nuclear protein detection - TLE4 shows B-cell-restricted expression in hematopoietic cells, making it valuable for B-cell lineage studies

• Neurological tissue studies - Due to TLE4's predominant expression in brain tissue, particularly in the caudate nucleus

• Muscle stem cell research - For investigating quiescence and skeletal muscle regeneration mechanisms

The choice of biotin conjugation is particularly valuable when high sensitivity is required or when using detection systems compatible with streptavidin-biotin interactions.

How does TLE4 expression vary across different tissue and cell types?

TLE4 demonstrates distinct expression patterns across various tissues and cellular contexts:

This expression pattern makes TLE4 antibodies particularly useful for studies involving B-cell development, brain tissue analysis, and muscle stem cell regulation.

How does the method of biotinylation affect TLE4 antibody specificity and performance?

The biotinylation method significantly impacts TLE4 antibody specificity and performance. Research has demonstrated:

N-hydroxysuccinimide-biotin (NHS-biotin) Method:

• Modifies primary amine groups on antibodies

• Can cause loss of antibody specificity

• May induce cross-reactivity with irrelevant antigens

• Likely alters the antigen-binding site structure

Biotin Hydrazide Method:

• Links biotin to the glycan moiety of antibodies

• Better preserves antibody specificity

• Recommended when high antigenic specificity is required

• Less likely to interfere with antigen recognition

This difference occurs because:
"Biotinylation of Mabs by the N-hydroxysuccinimide-biotin (NHS-biotin) resulted in crossreactivity of each Mab with irrelevant peptides. In contrast, Mab specificity was preserved after biotinylation with biotin hydrazide, which links biotin to the glycan moiety of antibodies."

For optimal results with biotin-conjugated TLE4 antibodies, researchers should:

• Verify the biotinylation method used by the manufacturer

• Consider biotin hydrazide-based conjugates for experiments requiring high specificity

• Include appropriate controls to account for potential non-specific binding

What are the molecular mechanisms through which TLE4 regulates transcriptional repression in different signaling pathways?

TLE4 functions as a sophisticated transcriptional corepressor through multiple mechanisms:

In Wnt Signaling:

• TLE4 antagonizes TCF/LEF-mediated target gene activation

• Competes with or modulates β-catenin's access to TCF factors on Wnt-responsive enhancers

• Is inactivated during Wnt signaling by ubiquitylation, specifically by the HECT E3 ligase Hyd/UBR5

• TLE3 and TLE4 together participate in diminishing canonical Wnt signaling activity

In Muscle Stem Cell Regulation:

• TLE4 represses Pax7-mediated Myf5 transcriptional activation

• Occupies the −111 kb Myf5 enhancer to maintain quiescence

• Must be downregulated for satellite cell activation and myogenic commitment

• "TLE4 downregulation is required for Myf5 activation and myogenic commitment"

General Repression Mechanisms:

• Promotes chromatin compaction

• Facilitates histone deacetylation

• Interacts with various DNA-binding proteins including HES (Hairy/Enhancer-of-Split), RUNX, and TCF

This multi-faceted role makes TLE4 antibodies valuable tools for investigating various aspects of transcriptional regulation in different cellular contexts.

How can researchers optimize detection of TLE4 protein complexes using biotin-conjugated antibodies?

Optimizing detection of TLE4 protein complexes requires careful consideration of several factors:

Proximity-Based Labeling Approaches:

• BioID techniques have been successfully used to map TLE4 interactions with TCF7L1 in mESCs

• This approach can identify both known partners (like β-catenin) and novel proximal proteins

• "Known TCF7L1 interactors TLE3/4 and β-catenin, and numerous proteins not previously associated with TCF7L1, were identified in both [BioID] systems"

Technical Optimization Steps:

• Background Control: Include controls for background biotinylation, as "streptavidin staining revealed a considerable amount of background biotinylation in wildtype mESCs, even in the absence of biotin"

• Co-IP Protocol Modifications:

  • For detecting TLE4-β-catenin complexes, stimulate cells with GSK-3 inhibitors (e.g., LiCl, CHIR99021)

  • Use gentler lysis conditions to preserve protein-protein interactions

  • Consider crosslinking to stabilize transient interactions

• Detection Systems:

  • For immunofluorescence: Use streptavidin conjugated to bright fluorophores

  • For western blotting: Employ streptavidin-HRP for enhanced sensitivity

  • For mass spectrometry: Apply streptavidin-biotin affinity purification followed by LC-MS/MS

Nuclear Localization Considerations:
TLE4 exhibits a "dense punctate pattern to the nuclei" of certain cell types, requiring specific nuclear preparation methods for optimal detection of protein complexes.

What controls should be included when using biotin-conjugated TLE4 antibodies in immunoassays?

Robust controls are essential for generating reliable data with biotin-conjugated TLE4 antibodies:

Essential Controls:

• Negative Controls:

  • Isotype control (biotin-conjugated antibody of the same isotype)

  • Secondary reagent only (streptavidin-reporter without primary antibody)

  • Samples known to be negative for TLE4 expression (e.g., T lymphocytes or myeloblasts)

  • "No labelling was observed in control experiments performed without incubation with the primary antibody"

• Positive Controls:

  • Cell lines with confirmed TLE4 expression (e.g., pro-B, pre-B, and mature B cells)

  • Recombinant TLE4 protein when available

  • Tissues with high TLE4 expression (brain caudate nucleus, pituitary gland, testis)

• Specificity Controls:

  • Competing with unconjugated TLE4 antibody

  • Preabsorption with recombinant TLE4 protein

  • Knockout/knockdown validation - "Clones of each gene knockout were sequenced to confirm and identify the genomic bi-allelic mutations"

• Biotinylation-Related Controls:

  • Endogenous biotin blocking reagents

  • Background assessment using streptavidin reagents on untreated samples

  • Comparison with non-biotinylated anti-TLE4 antibody

How can researchers apply biotin-conjugated TLE4 antibodies to investigate TLE4's role in muscle stem cell regulation?

Biotin-conjugated TLE4 antibodies can be valuable tools for studying TLE4's role in muscle stem cell biology:

Experimental Approaches:

• Temporal Expression Profiling:

  • Track TLE4 expression dynamics during muscle regeneration

  • "A subset of satellite cells transiently downregulate TLE4 during early time points following muscle injury"

  • Compare activated vs. quiescent satellite cell populations using biotin-TLE4 antibodies with fluorescent streptavidin detection

• Co-Localization Studies:

  • Use biotin-TLE4 antibodies with streptavidin-fluorophores alongside Pax7 antibodies

  • Investigate TLE4-Pax7 interaction at the -111 kb Myf5 enhancer

  • "TLE4 represses Pax7-mediated Myf5 transcriptional activation by occupying the −111 kb Myf5 enhancer to maintain quiescence"

• Functional Analysis:

  • Monitor TLE4 levels before and after satellite cell activation

  • Correlate TLE4 expression with Myf5 activation status

  • "Loss of TLE4 function causes Myf5 upregulation, an increase in satellite cell numbers and altered differentiation dynamics during regeneration"

• Methodological Approach:

  • Use biotin-conjugated TLE4 antibodies for ChIP assays to identify TLE4 binding sites

  • Employ proximity ligation assays (PLA) to confirm TLE4-Pax7 interactions in situ

  • Develop FACS protocols to isolate TLE4-high vs. TLE4-low satellite cell populations

This approach allows researchers to investigate the mechanistic details of how TLE4 maintains satellite cell quiescence and regulates muscle differentiation.

What are the key factors affecting the performance of biotin-conjugated TLE4 antibodies in different experimental applications?

Several factors critically influence the performance of biotin-conjugated TLE4 antibodies:

Application-Specific Considerations:

Host-Specific Considerations:
Rabbit polyclonal anti-TLE4 antibodies have been successfully used for various applications , but clone selection is crucial for specific applications.

Epitope-Related Factors:

• Antibodies targeting different regions of TLE4 may perform differently:

  • N-terminal region antibodies

  • Middle region antibodies

  • C-terminal region antibodies

• Choose epitopes that are accessible in your experimental conditions

Biotinylation Parameters:

• Biotin-to-antibody ratio affects performance

• Over-biotinylation can reduce antibody affinity

• Biotinylation method affects specificity - "Mab specificity was preserved after biotinylation with biotin hydrazide, which links biotin to the glycan moiety of antibodies"

Understanding these factors will help researchers select the most appropriate biotin-conjugated TLE4 antibody for their specific experimental needs and optimize protocols accordingly.

How can biotin-conjugated TLE4 antibodies be used to investigate Wnt signaling pathway dynamics?

Biotin-conjugated TLE4 antibodies provide valuable tools for investigating Wnt signaling dynamics:

Key Research Applications:

• Monitoring TLE4 Ubiquitylation:

  • Use biotin-conjugated TLE4 antibodies to detect changes in TLE4 levels during Wnt activation

  • "TLE is inactivated during Wnt signaling by Hyd/UBR5-dependent ubiquitylation"

  • Track TLE4 protein levels after Wnt3A treatment or GSK-3 inhibition

• TLE4-TCF Interaction Studies:

  • Investigate the dynamic relationship between TLE4 and TCF factors during Wnt activation

  • "β-catenin and TLE1 can bind simultaneously to TCF" - a similar mechanism may apply to TLE4

  • Use proximity ligation assays with biotin-TLE4 and TCF antibodies

• Chromatin Occupancy Dynamics:

  • Perform ChIP assays with biotin-TLE4 antibodies before and after Wnt activation

  • Use streptavidin pull-down approaches to identify DNA regions bound by TLE4

  • "Groucho/TLE is tethered to enhancers by DNA-binding proteins including HES, RUNX, and TCF and appears to repress transcription of linked genes primarily by chromatin compaction"

• Wnt-Regulated Gene Expression:

  • Correlate TLE4 chromatin occupancy with expression of Wnt target genes

  • "TLE3 and TLE4 participate in diminishing canonical Wnt signaling activity, supporting transcription of synaptic genes"

Experimental Design Considerations:

• Include both unstimulated and Wnt-stimulated conditions

• Employ GSK-3 inhibitors (CHIR99021, LiCl) to activate canonical Wnt signaling

• Compare results from biotin-conjugated TLE4 antibodies with other detection methods to confirm findings

What are the optimal protocols for detecting TLE4 in B-cell populations using biotin-conjugated antibodies?

Detecting TLE4 in B-cell populations requires specialized protocols due to its nuclear localization and B-cell-restricted expression in the hematopoietic system:

Optimized Protocol Elements:

• Sample Preparation:

  • For fixed cells: Use mild fixation (2-4% PFA, 10 minutes) followed by permeabilization

  • For nuclear preparations: "Immunostaining with the rabbit anti-TLE4 antibody revealed a dense punctate pattern to the nuclei of pro-B, pre-B and mature B cells"

  • Consider nuclear isolation techniques for highest specificity

• Staining Procedure:

  • Block endogenous biotin using avidin/biotin blocking kits

  • Include B-cell markers (CD19, CD20) for co-staining

  • Optimize antibody concentration (typically 1-5 μg/ml)

  • "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"

• Detection System:

  • For flow cytometry: Use streptavidin-PE or streptavidin-APC

  • For microscopy: Employ streptavidin-conjugated fluorophores with appropriate spectra

  • Consider signal amplification systems for low-abundance detection

• Validation Approach:

  • Use T-cells as negative controls: "No labelling was observed in control experiments performed without incubation with the primary antibody"

  • Include pro-B, pre-B, and mature B cells as positive controls

  • Confirm nuclear localization with DAPI co-staining

This protocol can be applied to study TLE4's role in B-cell development, differentiation, and function, leveraging its restricted expression pattern in the hematopoietic system.

How do different TLE family members interact, and how can biotin-conjugated antibodies help distinguish between them?

The TLE family includes several related proteins with distinct and overlapping functions, presenting challenges for specific detection:

TLE Family Interactions and Expression Patterns:

"TLE1 is upregulated in early differentiation stages while TLE2 and TLE4 are upregulated later, indicating roles in the transition of cells toward a terminally differentiated state"

Using Biotin-Conjugated Antibodies for Differentiation:

• Epitope Selection:

  • Use antibodies targeting unique regions of TLE4

  • Avoid conserved domains (WD40 repeats, Q domains) shared among TLE family

  • Validate specificity against other TLE proteins

• Sequential Immunoprecipitation:

  • Perform initial pull-down with biotin-conjugated TLE4 antibody

  • Use streptavidin beads for isolation

  • Follow with Western blot using antibodies specific for other TLE family members

  • This approach can identify TLE4-specific vs. shared complexes

• Multiplex Detection:

  • Combine biotin-conjugated TLE4 antibody with directly-labeled antibodies against other TLE proteins

  • Use different detection systems (e.g., streptavidin-Alexa647 for TLE4, direct fluorophores for others)

  • Analyze co-localization and relative expression levels

• Functional Validation:

  • "TLE3 and TLE4 participate in diminishing canonical Wnt signaling activity"

  • Design experiments to assess the specific contribution of TLE4 vs. other family members

  • Use biotin-conjugated antibodies for pull-down followed by functional assays

Understanding these interactions is crucial for interpreting experimental results and for designing studies that accurately distinguish the specific roles of TLE4 from other family members.