TCF15 Antibody, FITC conjugated

What is TCF15 and what is its biological significance in research?

TCF15 (Transcription Factor 15) is a basic helix-loop-helix (bHLH) transcription factor that plays crucial roles in developmental biology, particularly in stem cell differentiation. Research has identified TCF15 as a marker for differentiating pluripotent cells that are transitioning from naive to primed states. It is expressed in the inner cell mass of E4.5 blastocysts and in a subpopulation of embryonic stem cells (ESCs) that are primed for differentiation but still express Oct4 . TCF15 also has significant functions in hematopoiesis, where it is required for hematopoietic stem cell (HSC) quiescence and long-term self-renewal . Understanding TCF15 expression patterns can provide valuable insights into the mechanisms controlling the balance between pluripotency and lineage commitment.

What detection methods are compatible with TCF15 antibody (FITC conjugated)?

The FITC-conjugated TCF15 antibody (ABIN1940984) has been validated for enzyme-linked immunosorbent assay (ELISA) and Western Blotting (WB) applications . The fluorescent conjugation makes this antibody particularly suitable for flow cytometry, immunofluorescence microscopy, and other fluorescence-based detection methods. When using this antibody for Western blotting, researchers should expect to detect bands corresponding to the TCF15 protein (approximately 18-20 kDa). For quantitative applications such as flow cytometry, the FITC conjugation allows direct detection without secondary antibodies, simplifying experimental protocols and reducing background.

What is the binding specificity of this TCF15 antibody and how does it affect experimental design?

This particular TCF15 antibody recognizes amino acids 81-107 in the central region of human TCF15 . This specificity is important for experimental design as this region is relatively conserved but may differ between species. The antibody was generated using rabbits immunized with a KLH-conjugated synthetic peptide corresponding to this amino acid sequence . When designing experiments, researchers should consider that this antibody has been specifically validated for human samples, and cross-reactivity with other species should be empirically determined before use in comparative studies. Additionally, researchers should note that this binding region differs from the DNA-binding domain shared with Scleraxis (Scx) , which may affect interpretations when studying TCF15's transcriptional activity.

How should samples be prepared for optimal TCF15 detection?

For optimal detection of TCF15 using this FITC-conjugated antibody, sample preparation should be tailored to the specific application. For flow cytometry, cells should be fixed with 2-4% paraformaldehyde and permeabilized with 0.1-0.5% Triton X-100 or saponin buffer to allow antibody access to intracellular TCF15. For Western blotting, samples should be lysed in a buffer containing protease inhibitors to prevent degradation of TCF15. When studying embryonic stem cells, it's important to consider that TCF15 expression is heterogeneous and dynamically regulated by culture conditions . Samples cultured in LIF+FCS will show heterogeneous TCF15 expression, while those in 2i conditions (ERK and GSK3β inhibitors) will show reduced expression . For immunofluorescence, nuclear counterstaining is recommended since TCF15 functions as a transcription factor.

How can this antibody be used to study the role of TCF15 in pluripotency transitions?

The FITC-conjugated TCF15 antibody can be employed to track pluripotency transitions by identifying TCF15-expressing subpopulations within heterogeneous stem cell cultures. Research has demonstrated that TCF15 marks a subpopulation of ESCs that are primed for differentiation while still expressing Oct4, but have downregulated naive pluripotency markers such as Nanog and Klf4 . To study these transitions:

• Use fluorescence-activated cell sorting (FACS) to isolate TCF15-high and TCF15-low populations

• Perform co-staining with pluripotency markers (Oct4, Nanog, Klf4) to identify transitional states

• Compare gene expression profiles between sorted populations using qPCR or RNA-sequencing

• Track changes in TCF15 expression during differentiation protocols to identify critical transition points

This methodological approach can provide insights into the molecular mechanisms controlling the earliest stages of lineage commitment, particularly since TCF15 expression precedes downregulation of key pluripotency factors .

What methodological considerations exist when using TCF15 antibody for multi-parameter flow cytometry?

When incorporating FITC-conjugated TCF15 antibody into multi-parameter flow cytometry panels, several methodological considerations are critical:

• Spectral overlap: FITC (emission peak ~520nm) has potential overlap with PE and other green-yellow fluorophores. Proper compensation controls should be included.

• Fixation/permeabilization optimization: Since TCF15 is an intracellular transcription factor, fixation and permeabilization protocols need optimization to maintain surface marker epitopes while enabling intracellular antibody access.

• Panel design: When studying pluripotency transitions, combine TCF15-FITC with antibodies against:

  • Oct4 (expressed in all pluripotent cells)

  • Nanog and Klf4 (expressed predominantly in naive cells)

  • Epiblast markers (to identify primed states)

• Sequential staining: Consider performing surface marker staining before fixation/permeabilization for TCF15 detection

• Live/dead discrimination: Include viability dyes compatible with fixed cells to eliminate false positives

This approach allows simultaneous identification of various pluripotent subpopulations based on their developmental status, providing resolution beyond traditional bulk analyses .

How can TCF15 antibody be utilized to investigate Id-mediated inhibition in stem cell differentiation?

The FITC-conjugated TCF15 antibody can be instrumental in studying the regulatory relationship between TCF15 and inhibitor of DNA binding/differentiation (Id) proteins. Id proteins inhibit TCF15 activity by preventing its heterodimeric binding with E47, thereby maintaining pluripotency . Methodological approaches include:

• Immunoprecipitation experiments to detect physical interactions between TCF15 and Id proteins

• Comparative immunofluorescence or flow cytometry to quantify changes in TCF15 levels after modulating Id expression

• Chromatin immunoprecipitation (ChIP) assays to assess TCF15 binding to target promoters with and without Id protein inhibition

• Reporter assays using TCF15 target sequences to measure transcriptional activity when Id levels are manipulated

These experimental approaches can help elucidate how the balance between TCF15 and Id proteins controls the transition from pluripotency to lineage commitment. Research has shown that Id proteins maintain pluripotency by preventing sequential transitions toward differentiation, while TCF15 becomes active upon downregulation of Id, driving cells toward differentiation .

What methodology should be employed to study TCF15's role in hematopoietic stem cell biology?

The FITC-conjugated TCF15 antibody can be used to investigate TCF15's critical role in hematopoietic stem cell (HSC) quiescence and self-renewal. Research has demonstrated that TCF15 is required for maintaining HSC quiescence, and its loss leads to stem cell exhaustion and impaired long-term regenerative capacity . Recommended methodological approaches include:

• Flow cytometric analysis of TCF15 expression in different HSC compartments (LT-HSC, ST-HSC, MPPs)

• Cell cycle analysis combining TCF15 staining with Ki67/Hoechst or BrdU incorporation

• Transplantation assays comparing TCF15-high versus TCF15-low sorted populations for long-term engraftment potential

• Gene expression profiling of TCF15-expressing cells to identify downstream targets regulating quiescence

When interpreting results, researchers should consider that disrupting TCF15 abrogates long-term engraftment potential in secondary transplantation, indicating its essential role in maintaining stem cell function . The FITC conjugation enables live cell sorting of TCF15-expressing populations for functional studies and subsequent transplantation.

What controls and validation steps are necessary when studying TCF15 in differentiation experiments?

When using FITC-conjugated TCF15 antibody in differentiation studies, proper controls and validation are essential:

Additionally, when studying TCF15's role in differentiation, researchers should consider that Tcf15 expression shows a transient spike during early differentiation before declining . This dynamic expression pattern necessitates careful timing of sample collection and analysis to capture relevant transitions. Comparing results with established transcriptional targets of TCF15 can further validate experimental findings.

How can this antibody be integrated into lineage tracing experiments for developmental biology?

The FITC-conjugated TCF15 antibody can be incorporated into sophisticated lineage tracing experiments to track the fate of TCF15-expressing cells during development. Single-cell lineage tracing has revealed TCF15's significant role in determining cell fate decisions . Methodological approaches include:

• Combine immunofluorescence detection of TCF15 with genetic lineage tracing using Cre-loxP systems

• Employ TCF15 antibody staining in conjunction with cell surface markers for lineage identification

• Utilize the antibody in time-lapse imaging experiments to track TCF15-expressing cells in real-time

• Perform antibody-based cell sorting followed by single-cell RNA-seq to determine developmental trajectories

These approaches allow researchers to address questions about whether TCF15 expression predicts specific lineage outcomes and how the timing of TCF15 expression affects developmental potential. Research has demonstrated that TCF15-expressing cells in ESC cultures predominantly contribute to somatic lineages rather than primitive endoderm , making this methodology particularly valuable for studying early lineage segregation.

What are the considerations when using TCF15 antibody to study genetically modified stem cell models?

When applying FITC-conjugated TCF15 antibody to genetically modified stem cell models, several considerations are important:

• Epitope integrity: Ensure genetic modifications (especially near the central region of TCF15) do not affect the antibody's binding epitope (AA 81-107)

• Expression dynamics: Modified TCF15 constructs (e.g., Id-resistant forms) may show altered expression patterns compared to endogenous protein

• Autofluorescence: Some genetic modifications (especially those using fluorescent proteins) may interfere with FITC detection

• Cross-validation: Combine antibody detection with genetic reporters (e.g., TCF15-Venus) for robust signal verification

• Functional correlation: Correlate antibody-detected expression levels with functional readouts (differentiation speed, marker expression)

Research has utilized engineered forms of TCF15 that are tethered to E47 via a flexible linker, rendering them more resistant to Id inhibition . When studying such models, it's critical to verify that the antibody can still recognize the modified protein and to use appropriate controls to distinguish between endogenous and modified TCF15.

How should researchers interpret TCF15 detection patterns in relation to other pluripotency factors?

Interpreting TCF15 detection patterns requires consideration of its relationship with other pluripotency factors:

• TCF15 and Oct4 co-expression: TCF15-expressing cells maintain Oct4 expression (>95%), indicating they remain pluripotent despite being primed for differentiation

• TCF15 and Nanog/Klf4 exclusivity: Less than 15% of TCF15-high cells express Nanog or Klf4, suggesting TCF15 marks a population that has downregulated naive pluripotency markers

• Temporal dynamics: TCF15 expression increases as cells transition from naive to primed states, preceding lineage-specific marker expression

• Heterogeneity interpretation: Mosaic TCF15 expression within morphologically undifferentiated colonies indicates dynamic subpopulations within ESC cultures

When analyzing experimental results, researchers should consider that TCF15 marks a transitional state rather than a terminal differentiation outcome. Quantitative immunofluorescence or flow cytometry combining TCF15 with other pluripotency markers can provide insights into the hierarchical relationships between different pluripotent states and their developmental potentials.

What are common technical challenges when using TCF15 antibody and how can they be addressed?

Researchers frequently encounter several technical challenges when working with FITC-conjugated TCF15 antibody:

Additionally, researchers should note that TCF15 shows dynamic expression during differentiation, with a transient spike during early differentiation followed by downregulation . Timing sample collection appropriately is therefore critical for capturing relevant expression patterns.

How can quantitative analysis of TCF15 expression be optimized for reproducibility?

To ensure reproducible quantitative analysis of TCF15 expression:

• Standard curve calibration: Use quantitative flow cytometry with calibration beads to convert fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF)

• Reference standards: Include biological reference samples with known TCF15 expression levels in each experiment

• Normalization strategy: Normalize TCF15 expression to appropriate housekeeping genes or proteins when performing comparative analyses

• Consistent gating strategy: Develop and strictly adhere to standardized gating protocols for identifying TCF15-positive populations

• Environmental standardization: Control for culture conditions, cell density, and passage number, as these factors influence TCF15 expression

• Technical replicates: Include sufficient technical and biological replicates to account for the heterogeneity in TCF15 expression