TBX6 Antibody, FITC conjugated

What is TBX6 and why is it significant in developmental biology?

TBX6 is a T-box transcription factor that plays an essential role in determining the fate of axial stem cells, specifically in the neural versus mesodermal lineage decision. It acts partly by down-regulating a specific enhancer (N1) of SOX2 to inhibit neural development, while also playing critical roles in left/right axis determination through effects on Notch signaling and the morphology of nodal cilia .

In developmental contexts, TBX6 expression is temporally regulated - transient expression induces mesoderm and cardiovascular specification from pluripotent stem cells, while prolonged expression suppresses cardiac differentiation and promotes somite lineages including skeletal muscle and chondrocytes . This temporal regulation makes TBX6 a fascinating target for developmental biology research.

What are the primary applications for TBX6 antibodies in research?

TBX6 antibodies are valuable tools in multiple research applications including:

• Western blot detection (typically at 5 μg/mL concentration with HRP-conjugated secondary antibodies diluted 1:50,000-100,000)

• Immunocytochemistry/Immunofluorescence for examining TBX6 expression in cells and tissues

• ELISA assays (with dilutions of approximately 1:62500)

• Flow cytometry (particularly for FITC-conjugated antibodies)

• Detection of TBX6 in developmental studies of mesoderm formation and differentiation

These applications provide researchers with methods to investigate TBX6 expression, localization, and function in various experimental systems.

How do I select the appropriate TBX6 antibody for my research application?

Selection should be based on:

• Target species reactivity: Ensure the antibody recognizes TBX6 in your experimental species. Available antibodies react with human, mouse, and rat TBX6 .

• Application compatibility: Different antibody preparations perform optimally in specific applications:

  • For Western blot: Unconjugated polyclonal antibodies are often preferred

  • For immunofluorescence: FITC-conjugated antibodies eliminate the need for secondary antibody incubation

  • For multicolor flow cytometry: Consider fluorophore spectral properties to avoid overlap with other markers

• Clonality considerations: Polyclonal antibodies recognize multiple epitopes and may provide stronger signals, while monoclonal antibodies offer higher specificity .

• Validated applications: Review scientific literature and product documentation for validation in your specific application.

What are the optimal protocols for detecting TBX6 in differentiating stem cells using FITC-conjugated antibodies?

For detecting TBX6 in differentiating stem cells:

• Sample preparation:

  • Fix cells using 4% paraformaldehyde for 15-20 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 for 10 minutes

  • Block with 5% normal serum in PBS for 1 hour

• Antibody staining:

  • Use TBX6 antibody, FITC conjugated at 0.2-10 μg/mL (optimize for your specific antibody)

  • Incubate overnight at 4°C or 3 hours at room temperature in blocking solution

  • Counterstain nuclei with DAPI (1:1000)

• Visualization:

  • Image using epifluorescence or confocal microscopy

  • FITC is optimally excited at 494 nm and emits at 518 nm

The key to successful detection is timing, as TBX6 expression is dynamic during differentiation. In mouse ESC differentiation models, TBX6 expression peaks during the nascent mesoderm stage (approximately day 4 of differentiation) and is sharply downregulated thereafter .

How should Western blot protocols be optimized for detecting TBX6 protein?

For optimal Western blot detection of TBX6:

• Sample preparation:

  • Lyse cells in RIPA buffer with protease inhibitors

  • Load 20-40 μg total protein per lane

  • Use reducing conditions as demonstrated in R&D Systems' protocol

• Gel and transfer conditions:

  • Use 10-12% SDS-PAGE gels

  • Transfer to PVDF membrane (preferred over nitrocellulose for TBX6)

• Antibody incubation:

  • Block membrane with 5% non-fat dry milk in TBST

  • Incubate with primary TBX6 antibody at 5 μg/mL concentration

  • Use HRP-conjugated secondary antibody at 1:50,000-100,000 dilution

• Detection considerations:

  • Expected molecular weight: Primary band at approximately 40 kDa

  • Alternative band at 33-47 kDa may be observed due to isoforms or post-translational modifications

  • Use Immunoblot Buffer Group 3 as recommended for optimal results

This protocol has been verified for detecting TBX6 in HT1080 human fibrosarcoma and HL-60 human acute promyelocytic leukemia cell lines .

What methods are recommended for co-staining TBX6 with other mesoderm markers?

For effective co-staining of TBX6 with other mesoderm markers:

• Marker selection:

  • Nascent mesoderm: T/Brachyury, Mesp1, Eomes

  • Lateral/cardiac mesoderm: Flk1 and PDGFRα double-positive cells

  • Paraxial mesoderm: Msgn1, Meox1, Tcf15

  • Cardiac progenitors: Isl1, Nkx2.5, Gata4

• Antibody compatibility:

  • When using TBX6-FITC, select other primary antibodies raised in different host species

  • Use secondary antibodies with distinct fluorophores (e.g., Cy3, Cy5, or Alexa Fluor 647)

• Sequential staining protocol:

  • Incubate with unconjugated primary antibodies first

  • Add appropriate secondary antibodies

  • Finally add directly conjugated TBX6-FITC antibody

  • Include proper controls for each antibody

• Analysis considerations:

  • Use single-stained controls to set compensation in flow cytometry

  • For microscopy, capture single-channel images to assess potential bleed-through

This methodology has been demonstrated effective in studies examining T-GFP+ mesoderm populations co-stained with TBX6 and other lineage markers .

How do I quantitatively analyze TBX6 expression during mesoderm differentiation?

For quantitative analysis of TBX6 expression:

• qRT-PCR analysis:

  • Design primers specific to TBX6 (forward: 5'-AGGTTCTAGCAGCGAAGAGG-3', reverse: 5'-GTAGGATTGGTGCAACTCGG-3')

  • Normalize to stable reference genes (GAPDH, β-actin)

  • Track expression kinetics across differentiation timeline

  • Compare with other mesoderm markers (T, Mesp1) and neural markers (Sox2)

• Flow cytometry quantification:

  • Use FITC-conjugated TBX6 antibody alongside markers like T-GFP

  • Establish gates based on negative controls

  • Calculate percentage of single and double-positive populations

  • Track changes over differentiation timeline

• Western blot quantification:

  • Normalize band intensities to loading controls

  • Compare expression levels across differentiation timepoints

  • Generate relative expression curves

• Data representation:

  Differentiation Day	TBX6 Expression	T/Brachyury	Mesp1	Sox2
  Day 0-2	Low	Low	Low	High
  Day 3-4	Peak	Peak	Peak	Declining
  Day 5-6	Declining	Declining	Declining	Low
  Day 7+	Low	Low	Low	Low

This comprehensive approach allows tracking of TBX6 expression kinetics, which typically peaks during nascent mesoderm formation (around day 4 of differentiation) and sharply declines thereafter .

How can I interpret dual staining patterns of TBX6 with other developmental markers?

Interpreting dual staining patterns requires understanding the developmental context:

• TBX6 and T/Brachyury co-expression:

  • Strong co-expression indicates nascent mesoderm population

  • Cells expressing both markers represent an early mesodermal state

  • Quantify double-positive cells to assess mesoderm induction efficiency

• TBX6 and Sox2 relationship:

  • Typically mutually exclusive expression as TBX6 downregulates Sox2 enhancer

  • Any co-expressing cells may represent transitional states or neuromesodermal progenitors

  • High Sox2/low TBX6 indicates neural lineage commitment

• TBX6 with Flk1 and PDGFRα:

  • TBX6+/Flk1+/PDGFRα+ population represents lateral/cardiac mesoderm

  • TBX6+/Flk1-/PDGFRα+ cells typically represent paraxial mesoderm precursors

  • Changes in these populations over time reflect lineage specification

• Pattern analysis approach:

  • Create temporal maps of marker expression

  • Identify transition states where markers overlap temporarily

  • Correlate expression patterns with subsequent lineage commitment

Understanding these relationships helps decipher the complex process of mesoderm specification and subsequent lineage diversification regulated by TBX6.

How can TBX6 antibodies be used to investigate the temporal regulation of mesoderm lineage decisions?

TBX6 antibodies can provide crucial insights into temporal regulation of mesoderm differentiation:

• Pulse-chase experiments:

  • Use FITC-conjugated TBX6 antibodies to isolate cells at different TBX6 expression levels

  • Track fate of sorted populations to determine developmental trajectories

  • Correlate TBX6 expression duration with ultimate cell fate

• Live cell imaging protocols:

  • Use membrane-permeable fluorescent TBX6 antibody derivatives

  • Combine with reporter lines for other lineage markers

  • Track dynamic TBX6 expression and correlate with morphological changes

• Sequential sampling strategy:

  • Sample differentiating cultures at defined intervals (6-12 hours)

  • Perform immunostaining for TBX6 and lineage markers

  • Create temporal expression maps

Research has shown that transient TBX6 expression induces mesoderm and cardiovascular specification, while prolonged expression suppresses cardiac differentiation and induces somite lineages . These methodologies allow detailed investigation of these temporal effects.

What strategies can be used to investigate TBX6 target genes and regulatory networks?

To investigate TBX6 regulatory networks:

• ChIP-seq with TBX6 antibodies:

  • Use TBX6 antibodies for chromatin immunoprecipitation

  • Sequence bound DNA to identify genomic binding sites

  • Identify direct target genes and regulatory elements

  • Compare binding profiles at different developmental stages

• Combinatorial analysis approaches:

  • Integrate ChIP-seq with RNA-seq data

  • Identify genes whose expression changes correlate with TBX6 binding

  • Perform pathway analysis to identify enriched regulatory networks

• Verification methods:

  • Use reporter assays to confirm enhancer/promoter activity

  • Perform CRISPR/Cas9 editing of binding sites to validate functionality

  • Conduct co-immunoprecipitation to identify protein interaction partners

• Known regulatory relationships:

  • Direct upregulation of Mesp1

  • Repression of Sox2 enhancer (N1)

  • Interaction with BMP/Nodal/Wnt signaling pathways

These approaches can reveal how TBX6 orchestrates the complex gene regulatory networks governing mesoderm specification and differentiation.

How can TBX6 antibodies be used in single-cell analyses of developmental heterogeneity?

TBX6 antibodies enable sophisticated single-cell analyses:

• Single-cell protein detection methods:

  • Mass cytometry (CyTOF) incorporating TBX6 antibodies conjugated to metal isotopes

  • CITE-seq combining TBX6 antibody detection with transcriptome analysis

  • Imaging mass cytometry for spatial resolution of TBX6+ cells in tissues

• Computational analysis approaches:

  • Trajectory inference algorithms applied to TBX6 and lineage marker data

  • Pseudotime ordering of cells based on TBX6 expression levels

  • Branched differentiation path analysis to identify decision points

• Integration with transcriptomics:

  • Correlate TBX6 protein levels with single-cell RNA-seq data

  • Identify gene modules associated with different levels of TBX6

  • Define transcriptional states of TBX6-expressing cell populations

• Spatial context analysis:

  • Combine with tissue clearing techniques for whole-embryo imaging

  • Map TBX6+ cell locations relative to anatomical structures

  • Track spatial reorganization of TBX6+ cells during development

These methodologies have been validated in mouse embryo studies examining nascent mesoderm formation and have revealed critical insights into how TBX6 expression levels influence cell fate decisions .

How do I troubleshoot weak or absent TBX6 antibody staining in differentiation experiments?

When facing weak or absent TBX6 staining:

• Timing considerations:

  • TBX6 expression is highly dynamic and peaks at specific differentiation timepoints

  • Sample multiple timepoints, focusing on days 3-5 of differentiation when using mouse ESCs

  • Use T/Brachyury as a reference marker to identify nascent mesoderm stage

• Fixation optimization:

  • Test different fixation methods: 4% PFA (10-20 min), methanol (-20°C, 10 min)

  • Optimize permeabilization: 0.1-0.5% Triton X-100 or 0.1% Saponin

  • Consider antigen retrieval methods if working with tissue sections

• Antibody incubation parameters:

  • Test concentration range (2-15 μg/mL)

  • Compare overnight 4°C vs. room temperature incubation

  • Evaluate different blocking solutions (5% serum, 3% BSA, commercial blockers)

• Protocol verification:

  • Include positive control samples (e.g., embryonic mouse mesoderm, differentiated iPSCs)

  • Validate antibody performance with Western blot before immunostaining

  • Consider signal amplification systems for low abundance targets

These troubleshooting steps can help optimize detection of TBX6 protein in your experimental system.

What are the best methods for validating TBX6 antibody specificity?

To ensure TBX6 antibody specificity:

• Genetic validation approaches:

  • Test antibody on TBX6 knockout cell lines or tissues

  • Compare staining in wild-type versus TBX6-depleted samples using siRNA or CRISPR

  • Overexpress TBX6 and confirm increased signal

• Biochemical validation methods:

  • Perform peptide competition assays using the immunizing peptide

  • Test multiple antibodies targeting different TBX6 epitopes

  • Confirm band sizes on Western blot (expected: ~33-47 kDa)

• Expression pattern confirmation:

  • Verify that staining matches known expression patterns

  • Compare with mRNA expression by in situ hybridization

  • Confirm nuclear localization consistent with transcription factor function

• Cross-reactivity assessment:

  • Test reactivity with related T-box family members

  • Perform immunoprecipitation followed by mass spectrometry

  • Check specificity across multiple species if working in comparative models

These validation steps are essential to ensure that observed signals truly represent TBX6 protein and not artifacts or cross-reactive detection.