TCF7L1 Antibody, FITC conjugated

What is TCF7L1 and what cellular functions does it regulate?

TCF7L1 (also known as TCF3) is a member of the LEF/TCF (lymphoid enhancer factor/T cell factor) family of transcription factors that act as DNA binding partners for the WNT mediator β-catenin. The protein contains a highly conserved DNA binding high-mobility group (HMG)-box, a β-catenin binding domain in its amino terminus, and a Groucho/TLE binding domain in its central region . TCF7L1 functions in both WNT-dependent and WNT-independent pathways, playing crucial roles in development and disease processes. In canonical WNT signaling, TCF7L1 can partner with nuclear β-catenin to activate transcription of WNT target genes, while in the absence of WNT ligands, it binds to Groucho/TLE co-repressors to inhibit transcription .

What is the structure and specificity of the FITC-conjugated TCF7L1 antibody?

The FITC-conjugated TCF7L1 antibody (catalog No. ABIN7140179) is a rabbit polyclonal antibody specifically targeting amino acids 80-99 of the human TCF7L1 protein . It has been antigen affinity purified to enhance specificity and is of IgG isotype . The fluorescein isothiocyanate (FITC) conjugation enables direct visualization of TCF7L1 in fluorescence-based applications without requiring secondary antibody labeling. The antibody has been validated for human samples through various applications, though cross-reactivity with other species should be experimentally determined for each specific research context .

How do polyclonal TCF7L1 antibodies differ from monoclonal versions in research applications?

For TCF7L1 research specifically, polyclonal antibodies enable robust detection across various experimental conditions, particularly beneficial when studying protein variants or modified forms. Monoclonal antibodies, conversely, may offer higher specificity for discriminating between TCF7L1 and its closely related family members (TCF7L2, LEF1), which is essential when investigating paralog-specific functions in WNT signaling pathways .

What are the validated applications for FITC-conjugated TCF7L1 antibodies in cellular research?

The FITC-conjugated TCF7L1 antibody has been primarily validated for immunofluorescence applications, leveraging its direct fluorescent label for visualization of TCF7L1 localization within cells and tissues . While the specific product (ABIN7140179) requires additional application inquiries, similar TCF7L1 antibodies targeting comparable epitopes have demonstrated utility in:

• Immunofluorescence (IF) and immunocytochemistry (ICC) for cellular localization studies

• Western blotting (WB) for protein expression analysis

• ChIP and ChIP-seq for investigating TCF7L1 DNA binding sites

• CUT&RUN for high-resolution mapping of TCF7L1 genomic occupancy

For each application, optimization of antibody concentration, incubation conditions, and appropriate controls is essential for generating reliable data .

How should researchers design experiments to investigate TCF7L1's β-catenin-independent functions?

To investigate TCF7L1's β-catenin-independent functions, researchers should implement the following experimental design strategies:

• Utilize mutant TCF7L1 constructs: Generate or obtain TCF7L1 constructs with specific domain deletions, particularly TCF7L1ΔN (lacking the β-catenin binding domain) and TCF7L1ΔG (lacking the Groucho/TLE corepressor binding domain) .

• Implement parallel comparison experiments: Compare phenotypic outcomes between full-length TCF7L1, TCF7L1ΔN, TCF7L1ΔG, and TCF7L1* (DNA-binding deficient mutant) in relevant experimental models .

• Validate β-catenin independence: Perform co-immunoprecipitation assays to confirm the absence of β-catenin interaction with TCF7L1ΔN while maintaining interactions with other cofactors.

• Assess functional readouts: Measure parameters including cell proliferation (BrdU incorporation, Ki67 staining), migration capability, and senescence markers in response to expression of the different constructs .

Research has demonstrated that TCF7L1's tumor-promoting capabilities persist even when using the β-catenin binding-deficient TCF7L1ΔN mutant, suggesting a critical role for its repressor functions in tumorigenesis that are independent of canonical WNT/β-catenin signaling pathways .

What controls are essential when using FITC-conjugated TCF7L1 antibodies in fluorescence microscopy?

When conducting fluorescence microscopy with FITC-conjugated TCF7L1 antibodies, researchers must implement the following critical controls:

Essential controls:

• Isotype control: Use a FITC-conjugated rabbit IgG antibody (matching the host species and isotype of the TCF7L1 antibody) to assess non-specific binding and autofluorescence.

• Blocking peptide control: Pre-incubate the FITC-conjugated TCF7L1 antibody with the immunizing peptide (amino acids 80-99 of human TCF7L1) before staining to validate signal specificity .

• Knockdown/knockout validation: Include samples where TCF7L1 expression has been reduced or eliminated to confirm signal specificity.

• Cross-channel bleed-through control: When performing multi-color immunofluorescence, include single-stained controls to account for spectral overlap between fluorophores.

• Expression validation control: Correlate immunofluorescence findings with complementary techniques like western blotting to confirm expression patterns.

Additionally, researchers should optimize fixation methods, as TCF7L1 detection can be sensitive to overfixation with certain aldehydes, potentially masking the epitope recognized by the antibody .

How should researchers design experiments to investigate TCF7L1's role in tumor development models?

Based on established research methodologies, investigators studying TCF7L1's role in tumor development should implement a multi-faceted experimental approach:

• In vivo tumorigenesis models:

  • Chemical carcinogenesis protocols (e.g., DMBA/TPA skin carcinogenesis model) with TCF7L1 overexpression or knockdown

  • Xenograft models using human cancer cell lines with modulated TCF7L1 expression

  • Limiting dilution assays to assess tumor-initiating capacity

• Experimental design considerations:

  • Include appropriate genetic controls (littermates, empty vector controls)

  • Implement inducible expression systems (e.g., tet-inducible constructs) to regulate TCF7L1 expression timing

  • Monitor multiple parameters including tumor incidence, multiplicity, growth rate, and malignant conversion

  • Analyze both full-length TCF7L1 and domain-specific mutants to dissect mechanism dependencies

• Downstream analysis:

  • Histological assessment of tumor progression

  • Proliferation markers (BrdU incorporation, Ki67 staining)

  • Molecular profiling of tumor tissues

  • Identification of TCF7L1-dependent target genes through transcriptomic approaches

The research demonstrates that TCF7L1 overexpression significantly increases tumor incidence, multiplicity, and malignant conversion in skin carcinogenesis models, even in the absence of β-catenin binding capability, highlighting its potential role as a tumor promoter .

What mechanisms underlie TCF7L1's role in suppressing oncogene-induced senescence?

TCF7L1 has been identified as a suppressor of oncogene-induced senescence (OIS), a critical tumor-suppressive barrier that must be overcome during malignant transformation. The mechanisms through which TCF7L1 exerts this effect involve:

• Counteracting HRAS-induced senescence: Research demonstrates that TCF7L1 expression can suppress the senescence phenotype triggered by oncogenic HRAS expression .

• β-catenin independent activity: The senescence-suppressive function of TCF7L1 appears to be independent of β-catenin interaction, as TCF7L1ΔN (lacking β-catenin binding domain) maintains the ability to suppress senescence .

• Potential downstream mediators:

  • LCN2 (lipocalin 2) has been identified as a downstream effector of TCF7L1 that stimulates tumor growth .

  • TCF7L1 likely regulates additional genes involved in cell cycle checkpoint control and senescence pathway modulation.

• Repressor function relevance: TCF7L1's tumor-promoting and senescence-suppressing activities correlate with its function as a transcriptional repressor rather than its β-catenin-dependent activating capabilities, as evidenced by the reduced activity of the Groucho/TLE binding-deficient mutant (TCF7L1ΔG) .

Researchers investigating these mechanisms should employ senescence markers (SA-β-gal staining, p16/p21 expression, SASP factor analysis) and compare the effects of different TCF7L1 mutant constructs on senescence induction following oncogene activation .

How can researchers distinguish between TCF7L1 and other LEF/TCF family members in experimental settings?

Distinguishing between TCF7L1 and other LEF/TCF family members (TCF7L2, TCF7, LEF1) requires strategic experimental approaches:

• Antibody selection strategies:

  • Choose antibodies targeting non-conserved regions outside the highly conserved HMG-box DNA binding domain

  • The FITC-conjugated antibody targeting amino acids 80-99 provides specificity for TCF7L1 over other family members

  • Validate specificity through siRNA knockdown of individual family members

• Expression analysis techniques:

  • Paralog-specific qRT-PCR primers targeting unique exons or junctions

  • Western blotting exploiting size differences between family members

  • Isoform-specific RNA-seq analysis

• Functional discrimination approaches:

  • Selective knockdown/knockout studies targeting individual family members

  • Rescue experiments with specific family members in knockout backgrounds

  • ChIP-seq comparative analysis to identify unique and shared binding sites

• Domain-focused strategies:

  • Utilize the distinct protein interaction domains and unique post-translational modification sites

  • Exploit different binding affinities to β-catenin and Groucho/TLE co-repressors

When interpreting results, researchers should acknowledge the potential for functional redundancy between family members while identifying paralog-specific functions, particularly in contexts where multiple family members are co-expressed .

How should researchers optimize ChIP protocols specifically for TCF7L1 analysis?

Optimizing Chromatin Immunoprecipitation (ChIP) protocols for TCF7L1 requires careful consideration of several key parameters:

• Antibody selection considerations:

  • For FITC-conjugated antibodies: Consider using anti-FITC antibodies conjugated to beads for immunoprecipitation

  • Alternative approach: Use unconjugated TCF7L1 antibodies specifically validated for ChIP applications

  • Target epitopes outside DNA-binding domains to avoid interference with chromatin interaction

• Chromatin preparation optimization:

  • Crosslinking time: Optimize formaldehyde crosslinking (typically 10-15 minutes) to preserve TCF7L1-DNA interactions

  • Sonication parameters: Adjust to generate fragments of 200-500 bp for optimal resolution

  • Nuclear extraction conditions: Use gentle lysis buffers to preserve nuclear integrity

• Advanced protocol considerations:

  • Consider dual crosslinking (DSG followed by formaldehyde) for improved capture of protein-protein interactions

  • Implement epitope recovery steps (antigen retrieval) for certain fixation conditions

  • Use sequential ChIP (re-ChIP) to identify genomic regions bound by TCF7L1 and specific cofactors

• Controls and validation:

  • Include input controls, IgG controls, and positive controls (known TCF7L1 binding sites)

  • Validate findings with alternative TCF7L1 antibodies recognizing different epitopes

  • Consider parallel CUT&RUN approaches for complementary data generation

For high-quality TCF7L1 ChIP-seq datasets, assess enrichment at established TCF/LEF binding motifs and validate selected targets by ChIP-qPCR before proceeding to genome-wide analysis.

What are the optimal tissue fixation and antigen retrieval methods for detecting TCF7L1 in histological samples?

Optimal detection of TCF7L1 in histological samples requires careful selection of fixation and antigen retrieval methods:

Fixation protocols:

• Paraformaldehyde fixation:

  • 4% paraformaldehyde for 12-24 hours (tissue blocks)

  • 10-15 minutes for cultured cells or tissue sections

  • Buffer in neutral PBS to maintain epitope integrity

• Alternative fixatives:

  • Methanol/acetone (1:1) for 10 minutes at -20°C preserves nuclear antigens

  • Commercial fixatives optimized for transcription factor preservation

  • Avoid overfixation with glutaraldehyde which can mask nuclear epitopes

Antigen retrieval methods:

• Heat-induced epitope retrieval (HIER):

  • Citrate buffer (pH 6.0): 20 minutes at 95-98°C

  • EDTA buffer (pH 8.0-9.0): May provide superior results for nuclear transcription factors

  • Pressure cooker methods: 5 minutes at high pressure can improve detection

• Enzymatic retrieval:

  • Proteinase K treatment: Gentle digestion (1-5 μg/ml for 5-10 minutes)

  • Trypsin digestion: 0.05% at 37°C for 10-15 minutes

Optimization recommendations:

• Test multiple fixation and retrieval combinations in parallel on identical samples

• Include positive control tissues known to express TCF7L1

• Compare staining patterns with published TCF7L1 expression data

• Consider dual staining with β-catenin to confirm biological relevance of detected signal

Specific to FITC-conjugated TCF7L1 antibodies, researchers should be aware that some antigen retrieval methods may affect fluorophore stability, potentially requiring amplification steps using anti-FITC secondary antibodies .

How can researchers effectively study the interaction between TCF7L1 and its binding partners?

To effectively study interactions between TCF7L1 and its binding partners, researchers should implement a multi-technique approach:

• Co-immunoprecipitation (Co-IP) strategies:

  • Reciprocal Co-IP: Perform parallel experiments pulling down with TCF7L1 antibodies and partner protein antibodies

  • Nuclear extract preparation: Use gentle conditions to preserve protein-protein interactions

  • Crosslinking variants: Consider reversible crosslinkers for transient interactions

  • Antibody selection: Use antibodies targeting regions away from interaction domains

• Proximity ligation assay (PLA):

  • Enables visualization of protein interactions in situ with subcellular resolution

  • Particularly useful for examining β-catenin and Groucho/TLE interactions with TCF7L1

  • Quantifiable at single-cell level to assess interaction heterogeneity

• Domain mapping approaches:

  • Generate TCF7L1 constructs with domain deletions (TCF7L1ΔN, TCF7L1ΔG) to map interaction requirements

  • Perform mutational analysis of key residues within interaction interfaces

  • Use reporter assays to correlate binding with functional outcomes

• Advanced methodologies:

  • FRET/BRET analysis for real-time interaction dynamics in living cells

  • BioID or APEX proximity labeling to identify the TCF7L1 interaction network

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Functional validation:

  • Correlate interaction data with functional readouts (gene expression, cell behavior)

  • Use domain-specific mutants to establish causality between interactions and functions

  • Implement inducible systems to study temporal aspects of interaction dynamics

Research has established the importance of distinguishing between TCF7L1's interactions with β-catenin versus Groucho/TLE co-repressors, as these mediate distinct functional outcomes in contexts such as tumor promotion, where β-catenin-independent functions appear particularly significant .

How should researchers interpret conflicting data between TCF7L1 expression and WNT pathway activation?

When encountering conflicting data between TCF7L1 expression and WNT pathway activation, researchers should consider several key factors in their interpretation:

• Mechanistic considerations:

  • TCF7L1 functions in both β-catenin-dependent and -independent contexts

  • Context-dependent roles as both repressor (with Groucho/TLE) and activator (with β-catenin)

  • Potential feedback regulation within the WNT pathway affecting TCF7L1 levels

• Experimental reconciliation approaches:

  • Assess TCF7L1 post-translational modifications (phosphorylation status)

  • Evaluate subcellular localization of TCF7L1 and β-catenin

  • Examine concurrent expression of other LEF/TCF family members (potential redundancy)

  • Determine Groucho/TLE co-repressor availability in the system

• Contextual analysis framework:

  • Tissue/cell type specificity (differentiation state impacts WNT response)

  • Temporal dynamics (acute vs. chronic WNT activation)

  • Examination of TCF7L1 domains mediating effects (using domain mutants)

  • Analysis of downstream target genes beyond typical WNT targets

• Technical validation:

  • Confirm antibody specificity for detecting total vs. modified TCF7L1

  • Validate WNT activation using multiple readouts (TOPFlash, target gene expression)

  • Consider the impact of experimental manipulation timing on feedback mechanisms

Research has demonstrated that TCF7L1 can promote tumorigenesis independently of β-catenin interaction through its repressor function, which may explain apparent discrepancies between TCF7L1 expression and classical WNT pathway readouts .

What technical challenges might researchers encounter when using FITC-conjugated antibodies in multiplex immunofluorescence?

Researchers using FITC-conjugated TCF7L1 antibodies in multiplex immunofluorescence should anticipate and address several technical challenges:

• Spectral considerations:

  • FITC emission spectrum (peak ~525 nm) overlaps with other common fluorophores

  • Photobleaching susceptibility: FITC bleaches more rapidly than newer fluorophores

  • Autofluorescence interference: Tissues often exhibit background in the FITC channel

  • Solution: Implement proper spectral unmixing algorithms and sequential scanning

• Signal optimization challenges:

  • Signal amplification limitations: Direct conjugates lack secondary amplification

  • pH sensitivity: FITC fluorescence decreases significantly below pH 7.0

  • Fixative interactions: Some fixatives can quench FITC signal

  • Solution: Optimize fixation protocols and consider tyramide signal amplification

• Multiplexing-specific issues:

  • Antibody cross-reactivity: Validate each antibody combination

  • Uneven penetration: Ensure consistent distribution of all antibodies

  • Sequential staining interference: Earlier rounds may affect epitope availability

  • Solution: Test antibody compatibility and optimize staining sequence

• Tissue-specific considerations:

  • Nuclear antigen accessibility: TCF7L1 requires effective nuclear permeabilization

  • Lipofuscin autofluorescence: Particularly problematic in aged tissues

  • Varying expression levels: TCF7L1 may require different exposure settings than other targets

  • Solution: Implement tissue-specific permeabilization and autofluorescence quenching

• Data acquisition and analysis:

  • Dynamic range limitations: Balance settings for low and high expressors

  • Quantification challenges: Define appropriate nuclear segmentation parameters

  • Solution: Use appropriate controls for threshold setting and signal normalization

How can researchers distinguish authentic TCF7L1 signals from artifacts in their experimental systems?

Distinguishing authentic TCF7L1 signals from artifacts requires implementing rigorous validation strategies:

Biological validation approaches:

• Genetic manipulation controls:

  • siRNA/shRNA knockdown of TCF7L1 to confirm signal specificity

  • CRISPR/Cas9 knockout of TCF7L1 as definitive negative control

  • Rescue experiments with exogenous TCF7L1 expression in knockout backgrounds

• Expression pattern correlation:

  • Compare staining patterns with known TCF7L1 expression in reference tissues

  • Validate nuclear localization consistent with transcription factor function

  • Confirm expected changes during biological transitions (e.g., differentiation)

Technical validation strategies:

• Antibody validation:

  • Peptide competition assays using the immunizing peptide (AA 80-99)

  • Comparison of staining patterns using multiple TCF7L1 antibodies targeting different epitopes

  • Western blot confirmation of specificity at the expected molecular weight

• Signal authentication methods:

  • Secondary-only controls to detect non-specific secondary antibody binding

  • Isotype controls to assess non-specific binding of primary antibodies

  • Fluorophore-matched IgG controls to establish background thresholds

• Context-specific controls:

  • Technical replicate consistency assessment

  • Biological relevance confirmation (e.g., nuclear co-localization with DNA)

  • Expected biological correlations (e.g., relationship to β-catenin or WNT target gene expression)

Data interpretation guidelines:

• Require consistency across multiple detection methods

• Apply quantitative thresholds based on control samples

• Consider context-dependent variability in expression levels

• Interpret fluorescence intensity in relation to appropriate controls

• Document all validation steps performed when reporting results