TCF15 Antibody, Biotin conjugated

What is TCF15 and what are its primary biological functions?

TCF15, also known as Paraxis, is a basic helix-loop-helix transcription factor that plays multiple roles in developmental biology. It marks a subpopulation of pluripotent cells primed for somatic lineages and functions in early cell fate decisions. TCF15 expression is regulated by FGF signaling, and its activity is repressed by Id proteins, which are inhibitors of bHLH transcription factor activity . In early embryonic development, TCF15 is expressed in the inner cell mass of the E4.5 blastocyst, and it shows a distinct expression pattern that declines post-implantation before rising again at E8.5 during somite development . Recent research has also identified TCF15 as essential for hematopoietic stem cell (HSC) quiescence and long-term self-renewal .

How is a biotin-conjugated TCF15 antibody generated?

Biotin-conjugated TCF15 antibodies are typically generated through a multi-step process. First, rabbits are immunized with a KLH-conjugated synthetic peptide corresponding to amino acids within the TCF15 protein structure, commonly from regions such as amino acids 24-199 or 81-107 from the central region of human TCF15 . The antibodies are then purified through protein A columns, followed by peptide affinity purification to ensure specificity . The biotin conjugation process involves chemically linking biotin molecules to the purified antibody while maintaining its antigen-binding capacity. This creates a versatile research tool that can be used in conjunction with streptavidin-based detection systems to enhance sensitivity in various applications including ELISA and immunohistochemistry .

What are the advantages of using biotin-conjugated antibodies in TCF15 research?

Biotin-conjugated TCF15 antibodies offer several methodological advantages in research applications. First, the biotin-streptavidin interaction has one of the strongest non-covalent binding affinities known in biochemistry, providing exceptional sensitivity for detection of low-abundance TCF15 protein. Second, this conjugation system enables signal amplification through multiple biotin-streptavidin interactions, enhancing detection in samples where TCF15 expression may be limited, such as in subpopulations of pluripotent stem cells that are primed for differentiation . Third, biotin-conjugated antibodies provide versatility in experimental design as they can be paired with various streptavidin-conjugated reporter molecules (fluorophores, enzymes, quantum dots) without needing different primary antibodies. This is particularly valuable when working with heterogeneous cell populations expressing TCF15, such as the Venus-high versus Venus-low subpopulations observed in embryonic stem cell colonies .

How can researchers validate the specificity of biotin-conjugated TCF15 antibodies in pluripotent cell differentiation studies?

Validating the specificity of biotin-conjugated TCF15 antibodies requires a multi-faceted approach when studying pluripotent cell differentiation. First, researchers should perform antibody validation using positive and negative controls: positive controls can include samples with confirmed TCF15 expression (such as the Venus-high population in TCF15 reporter cell lines ), while negative controls should include samples where TCF15 is known to be absent or tissues from TCF15 knockout models. Second, epitope competition assays should be conducted where the antibody is pre-incubated with the immunizing peptide (AA 81-107 or other region-specific peptides) before application to samples; specific binding should be blocked by this competition . Third, researchers should perform parallel analyses with alternative detection methods, such as qPCR for TCF15 mRNA levels or using a second TCF15 antibody targeting a different epitope. This is particularly important when studying the transition of pluripotent cells from naive to primed states, as TCF15 expression dynamics are closely correlated with this transition .

What experimental approaches can be used to study the relationship between TCF15 and Id proteins using biotin-conjugated antibodies?

The relationship between TCF15 and Id proteins, which are inhibitors of bHLH transcription factor activity, can be studied using several experimental approaches with biotin-conjugated TCF15 antibodies. First, co-immunoprecipitation experiments can be performed where biotin-conjugated TCF15 antibodies are used to pull down TCF15 and associated proteins, followed by Western blot analysis for Id proteins . This approach can reveal direct interactions between TCF15 and Id proteins in various cellular contexts. Second, chromatin immunoprecipitation (ChIP) assays using biotin-conjugated TCF15 antibodies can identify genomic regions where TCF15 binds, and sequential ChIP (re-ChIP) can determine whether Id proteins co-occupy these regions. Third, researchers can employ proximity ligation assays (PLA) where biotin-conjugated TCF15 antibodies are used in conjunction with Id protein-specific antibodies to visualize direct interactions between these proteins at the single-molecule level within cells. These approaches are particularly relevant as research has shown that TCF15 activity is repressed by Id proteins and that TCF15 drives differentiation once released from Id inhibition .

How can biotin-conjugated TCF15 antibodies be utilized in single-cell analyses of hematopoietic stem cell populations?

Biotin-conjugated TCF15 antibodies offer powerful capabilities for single-cell analyses of hematopoietic stem cell (HSC) populations, particularly given TCF15's newly discovered role in HSC quiescence and long-term self-renewal . First, researchers can employ mass cytometry (CyTOF) using biotin-conjugated TCF15 antibodies followed by metal-tagged streptavidin, allowing simultaneous detection of TCF15 along with dozens of other markers to characterize heterogeneous HSC populations. Second, for multiplexed imaging approaches, biotin-conjugated TCF15 antibodies can be combined with cyclic immunofluorescence or imaging mass cytometry to spatially resolve TCF15 expression within the bone marrow niche. Third, for single-cell sorting applications, biotin-conjugated TCF15 antibodies with fluorescent streptavidin can identify TCF15-expressing cells for downstream applications like single-cell RNA sequencing or functional assays. This combined approach is particularly valuable when investigating the molecular mechanism by which TCF15 functions as a master regulator controlling HSC output, as suggested by CRISPR screening results showing TCF15 sgRNA had robust effects across biological replicates .

What protocol modifications are necessary when using biotin-conjugated TCF15 antibodies for immunohistochemistry of embryonic tissues?

When performing immunohistochemistry with biotin-conjugated TCF15 antibodies on embryonic tissues, several critical protocol modifications are necessary. First, endogenous biotin blocking is essential, particularly in embryonic tissues which often contain high levels of endogenous biotin. This can be achieved by pre-incubating sections with avidin followed by biotin before applying the biotin-conjugated TCF15 antibody. Second, antigen retrieval methods should be optimized specifically for TCF15 detection in embryonic tissues; heat-induced epitope retrieval in citrate buffer (pH 6.0) is often effective for accessing the TCF15 epitope without damaging the tissue architecture of delicate embryonic samples. Third, signal amplification systems should be carefully selected; using tyramide signal amplification can enhance detection sensitivity when visualizing TCF15 in the inner cell mass of E4.5 blastocysts where it is known to be expressed . Fourth, counterstaining should be performed with markers that help identify specific embryonic structures, such as Oct4 for pluripotent cells or Gata6 for primitive endoderm, enabling proper contextualization of TCF15 expression patterns throughout early development .

What is the optimal methodology for simultaneous detection of TCF15 protein and mRNA in differentiation studies?

For simultaneous detection of TCF15 protein and mRNA in differentiation studies, researchers should employ a combined immunofluorescence and RNA fluorescence in situ hybridization (IF-FISH) approach with specific optimizations. First, the RNA detection should precede protein detection, using RNAscope technology with probes targeting TCF15 mRNA followed by tyramide signal amplification to ensure signal retention through subsequent protein detection steps. Second, after RNA detection, biotin-conjugated TCF15 antibodies should be applied with a non-overlapping fluorophore-conjugated streptavidin to visualize the protein. Third, this dual detection should be performed at multiple timepoints during differentiation (0h, 24h, 48h, 72h) to capture the dynamic expression changes, as TCF15 shows a transient spike of expression during the early stages of ESC differentiation . Fourth, quantitative image analysis using specialized software should be employed to correlate mRNA and protein levels on a cell-by-cell basis, allowing researchers to determine whether the differentiation-primed state marked by TCF15 is regulated at the transcriptional or post-transcriptional level. This approach is particularly valuable when studying how TCF15 marks the transition from naive pluripotency to a differentiation-primed state .

How can biotin-conjugated TCF15 antibodies be integrated into ChIP-seq protocols to identify TCF15 transcriptional targets?

Integrating biotin-conjugated TCF15 antibodies into ChIP-seq protocols requires several strategic optimizations to identify TCF15 transcriptional targets. First, crosslinking conditions should be specifically optimized for TCF15; as a bHLH transcription factor, TCF15 may require dual crosslinking with both formaldehyde and protein-specific crosslinkers like DSG (disuccinimidyl glutarate) to efficiently capture transient DNA-protein interactions. Second, chromatin fragmentation should target fragments between 200-300bp, which is optimal for capturing binding sites of transcription factors like TCF15. Third, the immunoprecipitation step should utilize streptavidin-coated magnetic beads to capture the biotin-conjugated TCF15 antibody complexes, with specific attention to salt concentration in wash buffers to reduce background while maintaining specific interactions. Fourth, sequential ChIP (re-ChIP) can be performed to identify genomic regions co-occupied by TCF15 and its heterodimerization partner E47, as TCF15 has been studied in a tethered form with E47 that renders it more resistant to inhibition by Id proteins . This approach can help identify the direct transcriptional targets through which TCF15 represses Nanog and accelerates the transition of ESCs through the epiblast state while suppressing primitive endoderm differentiation .

How should researchers interpret conflicting data between TCF15 antibody detection and reporter systems?

When faced with discrepancies between biotin-conjugated TCF15 antibody detection and TCF15 reporter systems (such as the Tcf15-Venus reporter ), researchers should implement a systematic analytical approach. First, examine the temporal dynamics of the conflicting signals, as TCF15 protein may persist after mRNA levels have decreased, resulting in a temporal offset between reporter activity (which often reflects transcriptional activity) and antibody detection (which indicates protein presence). Second, consider epitope accessibility issues; the biotin-conjugated antibody targets a specific epitope (such as AA 81-107 ) that might undergo conformational changes or be masked by protein-protein interactions in certain cellular states, while reporter systems are independent of such constraints. Third, quantify the correlation between antibody and reporter signals across different cell populations and differentiation stages using flow cytometry or quantitative immunofluorescence, as was done for the Venus-high and Venus-low ESC populations . Fourth, validate findings using complementary techniques such as Western blotting for protein levels and qPCR for mRNA levels. This comprehensive approach will help determine whether discrepancies reflect biological reality or technical limitations.

What control experiments are necessary to distinguish between specific and non-specific binding in multiplexed detection systems using biotin-conjugated TCF15 antibodies?

In multiplexed detection systems using biotin-conjugated TCF15 antibodies, rigorous control experiments are essential to distinguish specific from non-specific binding. First, competitive blocking controls should be performed where excess free biotin is added prior to the addition of streptavidin-conjugated detection reagents to verify that signals arise from biotin-streptavidin interactions rather than non-specific binding. Second, isotype controls should be implemented using biotin-conjugated antibodies of the same isotype (Ig fraction from rabbit ) but targeting irrelevant antigens, processed identically to TCF15 antibodies. Third, absorption controls are critical, where biotin-conjugated TCF15 antibodies are pre-incubated with recombinant TCF15 protein before application to samples, which should abolish specific binding. Fourth, cross-reactivity assessment should be conducted by applying the detection system to samples known to lack TCF15 expression but expressing related bHLH factors like Scleraxis (Scx), which shares a nearly identical DNA binding domain with TCF15 . Fifth, multicolor controls in multiplexed systems should include single-antibody staining to establish spectral unmixing parameters and verify signal specificity in each channel independently.

How can researchers differentiate between TCF15 detection in pluripotent versus differentiation-primed cell states?

Differentiating TCF15 detection between naive pluripotent and differentiation-primed cell states requires a nuanced experimental approach. First, researchers should employ co-staining with established markers of naive pluripotency (Nanog, Klf4) and differentiation-primed states (FGF5, Otx2) alongside biotin-conjugated TCF15 antibodies, as studies have shown that less than 15% of TCF15-Venus-high cells express Nanog or Klf4 whereas over 95% express Oct4 . Second, culture conditions should be systematically varied between those that support naive pluripotency (2i/LIF) and those that promote differentiation priming (FGF2/activin), followed by quantitative analysis of TCF15 protein levels. Third, single-cell correlation analysis should be performed to determine how TCF15 levels fluctuate relative to other markers during the transition from naive to primed states. Fourth, functional assays should be conducted by sorting cells based on TCF15 antibody binding intensity and assessing their differentiation kinetics and lineage potential. This approach mirrors previous research showing that Tcf15 expression is regulated by FGF signaling and is particularly associated with cells transitioning from naive to epiblast-primed states .

What are the most promising applications of TCF15 antibodies in single-cell lineage tracing studies?

Biotin-conjugated TCF15 antibodies hold considerable promise for advancing single-cell lineage tracing studies in several key areas. First, these antibodies can enable prospective isolation of TCF15-expressing cells for subsequent transplantation experiments and long-term lineage tracing, particularly valuable in light of TCF15's newly discovered role in hematopoietic stem cell quiescence and long-term self-renewal . Second, they can be incorporated into sequential protein profiling methods on fixed tissue sections, allowing researchers to retrospectively map the spatial distribution of TCF15-expressing cells and their progeny throughout development or disease progression. Third, integration with genetic lineage tracing tools is possible by using biotin-conjugated TCF15 antibodies to validate the specificity of Tcf15-Cre or Tcf15-CreERT2 driver lines, ensuring accurate fate mapping of cells that express TCF15 at specific developmental timepoints. Fourth, these antibodies can be used in combination with single-cell RNA sequencing to correlate TCF15 protein levels with transcriptional states during cell fate transitions. This application is particularly relevant given that Tcf15 expression is regulated by FGF signaling and marks a subpopulation of epiblast-primed Oct4-positive cells .

How might TCF15 antibodies contribute to understanding the molecular mechanisms of lineage priming in pluripotent stem cells?

Biotin-conjugated TCF15 antibodies can significantly advance our understanding of lineage priming mechanisms in pluripotent stem cells through several methodological approaches. First, these antibodies enable the physical isolation of TCF15-expressing subpopulations for comprehensive multi-omics analysis (transcriptomics, proteomics, epigenomics), allowing researchers to characterize the molecular signature of differentiation-primed pluripotent cells. Second, they facilitate real-time tracking of TCF15 protein dynamics during differentiation using live-cell imaging with streptavidin-conjugated fluorophores, providing insights into how quickly cells transition from a TCF15-negative to TCF15-positive state. Third, TCF15 antibodies can be used in proximity-based labeling approaches (BioID or APEX) to identify the protein interaction network of TCF15 in pluripotent versus differentiation-primed states, potentially revealing how TCF15 coordinates with other factors to drive differentiation. Fourth, these antibodies enable functional validation studies where TCF15-high and TCF15-low populations are separately exposed to differentiation stimuli to quantify their differentiation kinetics and lineage bias, building upon earlier findings that an Id-resistant form of TCF15 rapidly downregulates Nanog and accelerates the transition of ESCs through the epiblast state .

What role might TCF15 antibodies play in developing new therapeutic approaches for hematopoietic disorders?

Biotin-conjugated TCF15 antibodies could contribute significantly to developing novel therapeutic approaches for hematopoietic disorders based on recent discoveries about TCF15's role in hematopoiesis. First, these antibodies can be used to identify and isolate TCF15-expressing hematopoietic stem cells (HSCs) with enhanced quiescence and long-term self-renewal properties , potentially providing a superior cell population for bone marrow transplantation therapies. Second, they enable high-throughput screening platforms to identify small molecules that modulate TCF15 expression or activity in HSCs, potentially yielding new chemical entities that could enhance HSC expansion ex vivo or improve engraftment in vivo. Third, TCF15 antibodies can facilitate the development of targeted delivery systems (such as antibody-drug conjugates) that specifically act on TCF15-expressing cells to modulate their function in hematological malignancies where stem cell dysfunction plays a role. Fourth, these antibodies can be used to monitor TCF15 expression as a biomarker for HSC functionality in patients undergoing treatment for hematological disorders, potentially providing prognostic information and guiding therapeutic decisions. This approach builds upon research identifying TCF15 as a master regulatory factor in the molecular program controlling HSC output through CRISPR screening .