TCF7L1 Antibody

What is TCF7L1 and why is it significant in cancer research?

TCF7L1 (also known as TCF3) is a member of the TCF/LEF family of transcription factors that functions predominantly as a transcriptional repressor in the Wnt/β-catenin signaling pathway. TCF7L1 has emerged as a significant factor in cancer research due to its oncogenic role in colorectal cancer (CRC), where it promotes cell migration, invasion, and adhesion by repressing growth arrest specific 1 (GAS1) expression . Notably, patients with elevated TCF7L1 expression have been shown to have poorer clinical outcomes, with a median survival of just 47 months compared to 93 months in patients with unaltered expression .

What are the structural characteristics of TCF7L1 protein that antibodies can target?

Human TCF7L1 is a 63 kDa protein, 588 amino acids in length, containing one HMG box DNA-binding domain (amino acids 346-414) . Most commercially available antibodies target specific regions, such as the C-terminal region (Lys429-Ser581) or the HMG box domain. Understanding these structural characteristics is crucial when selecting antibodies for specific experiments, particularly when studying protein-protein interactions or DNA binding activities .

How do I validate TCF7L1 antibody specificity for my experimental system?

Validation should involve multiple approaches:

• Western blot analysis showing the expected molecular weight band (~70 kDa) in relevant tissues (e.g., pancreas, lung, spleen)

• Positive controls using tissues with known TCF7L1 expression (e.g., human embryonic stem cells, hair follicles)

• Negative controls using TCF7L1 knockout or knockdown systems

• Cross-reactivity assessment with other TCF family members (TCF-4, TCF-12)

For comprehensive validation, immunoprecipitation followed by mass spectrometry can confirm antibody specificity beyond traditional methods .

What are the optimal applications for TCF7L1 antibodies in cancer research?

Based on published research, TCF7L1 antibodies have been successfully employed in:

How can I optimize TCF7L1 antibody use in ChIP-seq experiments to identify direct binding targets?

For optimal ChIP-seq experiments with TCF7L1 antibodies:

• Validate antibody specificity using known TCF7L1 binding sites (e.g., MYC 3' WRE and DKK4 promoter) by ChIP-qPCR before proceeding to sequencing

• Use crosslinking with 1% formaldehyde for 10 minutes at room temperature

• Consider using epitope-tagged (FLAG-tagged) TCF7L1 for enhanced specificity and reduced background

• Include appropriate controls, such as TCF7L1 mutants with disrupted DNA-binding capacity (e.g., lysine to proline substitution at residue 387 and proline to lysine substitution at residue 411)

• For data analysis, focus on binding regions within 2.5 kb of the transcription start site (TSS) of protein-coding genes to identify direct regulatory targets

What methods can be used to study TCF7L1's role in cell migration and invasion?

Based on published research, multiple complementary assays can be employed:

• Scratch-wound healing assays: Monitor wound closure at 0 and 24 hours in control vs. TCF7L1-manipulated cells

• Transwell migration assays: Seed cells (1 × 10⁴ per well) in serum-free media in the upper chambers with 10% FBS in lower chambers; assess after 48 hours

• Transwell invasion assays: Similar to migration assays but using Matrigel-coated membranes

• Adhesion assays: Seed cells (1 × 10³ per well) on collagen I-coated plates (40 μg/ml); quantify adherent cells after 1 hour

All assays should include appropriate controls, such as TCF7L1 knockdown, overexpression, and rescue experiments with GAS1 .

How do I address non-specific binding when using TCF7L1 antibodies?

TCF7L1 antibodies may show 5-10% cross-reactivity with other TCF family members like TCF-4 and TCF-12 due to conserved domains . To minimize non-specific binding:

• Use optimal antibody dilutions determined through titration experiments

• Increase blocking time (5% BSA or milk in TBST for 1-2 hours)

• Include appropriate controls, such as TCF7L1 knockout or knockdown samples

• For highly specific detection, consider using antibodies targeting unique regions of TCF7L1 rather than conserved domains

• Validate results with multiple independent antibodies targeting different epitopes

What are the best storage and reconstitution practices for TCF7L1 antibodies?

For optimal performance of TCF7L1 antibodies:

• Reconstitute lyophilized antibodies at 0.5 mg/mL in sterile PBS

• Store reconstituted antibodies at 2-8°C for up to 1 month or aliquot and store at -20 to -70°C for up to 6 months

• Avoid repeated freeze-thaw cycles by preparing small aliquots

• Use a manual defrost freezer for long-term storage

• Before use, centrifuge the antibody solution briefly to collect all material at the bottom of the tube

How do I analyze TCF7L1 ChIP-seq data to identify direct target genes in cancer cells?

To accurately identify direct TCF7L1 target genes:

• Integrate RNA-seq and ChIP-seq datasets by overlapping differentially expressed genes with TCF7L1 binding sites

• Focus on genes with TCF7L1 binding sites within 2.5 kb of the transcription start site (TSS)

• Apply conservative analysis parameters to identify high-confidence targets (e.g., the study by Nature Scientific Reports identified 41 high-confidence direct targets)

• Perform motif analysis to confirm TCF7L1 binding motifs within the enriched regions

• Validate selected targets using RT-qPCR and functional assays

• Consider gene set overlap analysis to identify targets associated with specific cellular processes (e.g., EMT, migration)

How can I distinguish between TCF7L1's direct repressive effects and indirect regulatory impacts?

Distinguishing direct from indirect effects requires:

• Combining ChIP-seq data with transcriptome analysis (RNA-seq) following TCF7L1 manipulation

• Including DNA-binding deficient TCF7L1 mutants as controls to identify binding-dependent regulation

• Performing time-course experiments following TCF7L1 knockdown or overexpression to identify immediate versus delayed gene expression changes

• Using rescue experiments with potential target genes (e.g., GAS1) to validate direct regulatory relationships

• Employing reporter assays with wild-type and mutated TCF7L1 binding sites in target gene promoters

How can TCF7L1 antibodies be used to investigate interactions with other transcription factors in the Wnt pathway?

Advanced techniques include:

• Co-immunoprecipitation (Co-IP) using TCF7L1 antibodies followed by Western blot analysis for potential partners (β-catenin, TLE/Groucho)

• Sequential ChIP (Re-ChIP) to identify genomic loci co-occupied by TCF7L1 and other factors

• Proximity ligation assay (PLA) to visualize in situ protein-protein interactions

• RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) to identify novel TCF7L1 interaction partners

• Comparative ChIP-seq analysis of TCF7L1 and other TCF family members (TCF7, TCF7L2) to identify unique and shared binding sites

What methodological approaches can assess TCF7L1's role in cancer stem cell maintenance and differentiation?

Based on research findings:

• Sphere formation assays to assess self-renewal capacity in TCF7L1-modulated cancer cells

• Flow cytometry analysis of cancer stem cell (CSC) markers in TCF7L1 knockdown or overexpressing cells

• In vivo limiting dilution assays to quantify tumor-initiating cell frequency

• Single-cell RNA-seq to characterize heterogeneity in TCF7L1 expression and its correlation with stemness signatures

• Lineage tracing experiments in mouse models with conditional TCF7L1 knockout/overexpression

How can TCF7L1 antibodies be used to develop potential diagnostic or prognostic tools for colorectal cancer?

Translational applications include:

• Immunohistochemical analysis of TCF7L1 expression in patient tissue microarrays, correlating with clinical outcomes

• Development of multiplexed immunofluorescence panels combining TCF7L1 with other prognostic markers

• Liquid biopsy approaches to detect circulating tumor cells with high TCF7L1 expression

• Correlation of TCF7L1 levels with response to specific therapies, especially those targeting Wnt signaling

• Development of TCF7L1-based gene expression signatures for patient stratification

What are promising approaches to target the TCF7L1-mediated transcriptional repression in cancer therapy?

Innovative strategies include:

• Development of small molecule inhibitors that disrupt TCF7L1-corepressor interactions

• Design of decoy oligonucleotides mimicking TCF7L1 binding sites to sequester the protein

• PROTAC (PROteolysis TArgeting Chimera) approaches for selective TCF7L1 degradation

• Targeting downstream effectors of TCF7L1 repression, such as GAS1

• Combination therapies targeting both TCF7L1 and other Wnt pathway components

How can new antibody technologies enhance TCF7L1 research beyond conventional applications?

Emerging technologies include:

• Development of recombinant antibody fragments (Fab, scFv) for improved tissue penetration and reduced background

• Engineered bispecific antibodies targeting TCF7L1 and its binding partners simultaneously

• Application of intrabodies to track and manipulate TCF7L1 in living cells

• Nanobody-based approaches for super-resolution imaging of TCF7L1 localization and dynamics

• Development of antibody-drug conjugates for targeted delivery to TCF7L1-expressing cancer cells