TBX6 Antibody, Biotin conjugated

What is TBX6 and what is its role in development?

TBX6 is a 47 kDa member of the T-box family of transcriptional regulators that plays essential roles during embryogenesis. It functions primarily in presomitic mesoderm development and promotes delta-like 1 transcription. Human TBX6 is 436 amino acids in length and contains one T-box DNA-binding domain (amino acids 90-277) . It acts as a critical determinant in the specification of mesodermal versus neural fate in developing embryos, partly by downregulating specific enhancers of SOX2 to inhibit neural development . TBX6 is also implicated in left/right axis determination through effects on Notch signaling around the node and influences on nodal cilia morphology and motility .

How does TBX6 influence mesoderm development specifically?

TBX6 functions as a key inducer of nascent mesoderm from pluripotent stem cells and plays a deterministic role in the specification of cardiovascular and somite lineages. Single-cell RNA-seq studies in mouse embryos and directed cardiac differentiation experiments in pluripotent stem cells have demonstrated that TBX6 induces mesoderm formation through direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling pathways . The temporal expression pattern of TBX6 is particularly important, as it dictates the developmental trajectory of mesodermal precursors toward specific lineages. During directed cardiac differentiation, TBX6 expression is rapidly induced at the mesoderm stage, peaks, and is then sharply downregulated, following kinetics similar to other nascent mesodermal genes .

What applications are TBX6 antibodies validated for?

Most commercially available TBX6 antibodies have been validated for the following applications:

These applications have been tested under specific conditions, and the antibody shows reactivity with both human and mouse TBX6, with human TBX6 showing approximately 90% amino acid identity with mouse TBX6 over amino acids 1-280 .

What are optimal fixation and staining protocols for TBX6 detection?

For immunohistochemistry of frozen tissue sections, immersion fixation has been successfully employed for embryonic mouse mesoderm (E9.5) with TBX6 antibody concentrations of approximately 10 μg/mL and overnight incubation at 4°C . For immunocytochemistry of stem cells, immersion fixation followed by incubation with 0.2 μg/mL antibody for 3 hours at room temperature has yielded specific nuclear staining in mesodermal cells . The protocol typically involves:

• Fixation of tissue/cells by immersion in appropriate fixative

• Blocking of non-specific binding sites

• Incubation with primary TBX6 antibody at optimized concentration

• Washing steps to remove unbound antibody

• Detection with fluorescently-labeled secondary antibody (e.g., NorthernLights 557-conjugated anti-goat IgG)

• Nuclear counterstaining with DAPI

• Mounting and imaging

When detecting biotin-conjugated antibodies, researchers should substitute steps 4-5 with streptavidin-conjugated detection reagents for optimal results.

How should I optimize Western blot conditions for TBX6 detection?

For Western blot applications, the following protocol has been successfully employed:

• Prepare protein lysates from appropriate cell lines (e.g., HT1080 or HL-60)

• Separate proteins on SDS-PAGE and transfer to PVDF membrane

• Block membrane with appropriate blocking buffer

• Incubate with TBX6 antibody at 2 μg/mL

• Wash thoroughly to remove unbound antibody

• Incubate with HRP-conjugated secondary antibody

• Visualize using appropriate chemiluminescence detection system

TBX6 typically appears as a specific band at approximately 40 kDa under reducing conditions . Optimal results have been obtained using specific immunoblot buffer systems (e.g., Immunoblot Buffer Group 3 as mentioned in the search results).

How can TBX6 antibodies be used to study mesoderm specification during development?

TBX6 antibodies can be employed to track mesoderm specification through immunostaining of developing embryos or differentiating stem cells. Research has shown that TBX6 is specifically expressed in the presomitic mesoderm during embryogenesis and in mesoderm-differentiated human iPSCs, with nuclear localization correlating with its active state . To effectively study this process, researchers can:

• Use TBX6 antibodies in combination with other mesoderm markers to identify specific mesoderm subtypes

• Perform time-course analyses during differentiation protocols to track the temporal dynamics of TBX6 expression

• Combine immunostaining with reporter systems (such as T-GFP) to monitor mesoderm induction in real-time

• Analyze subcellular localization to correlate nuclear TBX6 with active transcriptional states

Studies have demonstrated that TBX6 is rapidly induced during mesoderm formation, peaks during the nascent mesoderm stage, and is then downregulated as cells differentiate further, making it a valuable marker for early mesoderm specification events .

What controls should be included when using TBX6 antibodies in developmental studies?

Proper experimental controls are essential for interpreting TBX6 antibody staining results:

CRISPR/Cas9 technology has been successfully employed to generate TBX6 knockout embryonic stem cells, which serve as excellent negative controls for antibody validation. These knockout models have confirmed the critical role of TBX6 in mesoderm and cardiovascular differentiation, as its absence inhibits these developmental processes .

How can I validate TBX6 antibody specificity in my experimental system?

Validation of TBX6 antibody specificity can be accomplished through several approaches:

• Genetic validation: Use CRISPR/Cas9-mediated knockout of TBX6 as described in published research. Guide RNA targeted to the first exon of TBX6 can effectively eliminate expression, providing a clean negative control .

• Expression validation: Compare staining patterns between tissues or cells known to express TBX6 (e.g., presomitic mesoderm, differentiated mesoderm from iPSCs) versus those that do not express TBX6 (e.g., undifferentiated stem cells).

• Western blot validation: Confirm antibody detects a single band of the expected molecular weight (approximately 40 kDa) .

• Spatial-temporal validation: Verify that the expression pattern matches known developmental dynamics of TBX6, with expression peaking during mesoderm formation and declining thereafter.

How does TBX6 expression correlate with differentiation status in stem cells?

TBX6 expression serves as a valuable marker for mesoderm specification in differentiating stem cells. Immunostaining studies have revealed that TBX6 is absent in undifferentiated human iPSCs but shows strong nuclear localization upon differentiation into mesoderm . This pattern correlates with the functional role of TBX6 in driving mesoderm specification.

The temporal expression profile of TBX6 during directed cardiac differentiation follows a characteristic pattern:

• Absent in undifferentiated pluripotent stem cells

• Rapidly induced during initial mesoderm specification

• Peaks during the nascent mesoderm stage

• Sharply downregulated as cells commit to specific mesodermal lineages

This dynamic expression profile makes TBX6 an excellent marker for monitoring the precise timing of mesoderm induction and downstream lineage commitment events .

What are the key considerations when analyzing TBX6 expression in embryonic development?

When analyzing TBX6 expression during embryonic development, researchers should consider:

• Spatial restriction: TBX6 expression is normally confined to specific embryonic regions, particularly the presomitic mesoderm. Immunohistochemistry studies of embryonic mouse mesoderm (E9.5) have confirmed this localized expression pattern .

• Temporal dynamics: TBX6 expression follows precise temporal patterns during development, with expression peaks that correspond to critical developmental transitions.

• Subcellular localization: Active TBX6 shows nuclear localization, which correlates with its function as a transcription factor. The nuclear-to-cytoplasmic ratio can provide insights into its activation state.

• Co-expression with other markers: Analyzing TBX6 expression alongside other developmental markers can provide context for understanding its role in lineage specification.

• Genetic background effects: TBX6 expression patterns may vary depending on genetic background, which should be considered when comparing results across different model systems.

How can TBX6 antibody staining be integrated with functional studies?

Integrating TBX6 antibody staining with functional studies provides deeper insights into developmental processes:

• Combine with genetic perturbation: Use TBX6 antibody staining to assess the effects of CRISPR/Cas9 knockout or overexpression on mesoderm development. Research has demonstrated that TBX6 knockout in mouse pluripotent stem cells inhibits mesoderm and cardiovascular differentiation, while transient TBX6 expression induces mesoderm and cardiovascular specification .

• Correlate with signaling pathway analysis: TBX6 functions through activation of BMP/Nodal/Wnt signaling pathways. Combining TBX6 immunostaining with analysis of these pathway components can reveal functional relationships.

• Link with lineage tracing: TBX6 antibody staining can be combined with genetic lineage tracing approaches to track the fate of TBX6-expressing cells during development.

• Integrate with transcriptional profiling: Correlate TBX6 protein expression with transcriptomic data to understand its downstream effects on gene expression programs.

What are common issues when using TBX6 antibodies and how can they be resolved?

How can I enhance detection sensitivity for low-abundance TBX6 expression?

For detecting low levels of TBX6 expression, consider these approaches:

• Signal amplification: For biotin-conjugated antibodies, employ multiple layers of streptavidin-based amplification systems.

• Optimized fixation: Different fixation methods can significantly affect epitope accessibility. Testing multiple fixation protocols may identify conditions that better preserve the TBX6 epitope.

• Extended incubation: Longer primary antibody incubation times (e.g., overnight at 4°C) can improve detection of low-abundance targets.

• Sensitive detection systems: Use high-sensitivity detection reagents and imaging systems with appropriate dynamic range.

• Sample enrichment: When possible, enrich for TBX6-expressing cell populations before analysis.