TBX5 Antibody

What is TBX5 and why is it significant in cardiac and limb development research?

TBX5 is a 64 kDa member of the T-box family of transcription factors that plays a critical role in the development of the heart and upper limbs. The protein contains a conserved T-box DNA-binding domain (amino acids 58-238), a transactivation motif (Glu349-Glu-Asp351), and a nuclear localization signal (amino acids 339-379) . TBX5 is expressed in both embryonic and adult tissues, particularly in the heart, where it regulates the expression of genes such as FGF-10 and NPPA/ANF .

TBX5 is significant in developmental biology research because:

• It regulates transcription of several genes critical for proper heart morphogenesis

• It interacts with other transcription factors like NKX2.5 to synergistically activate cardiac-specific genes

• Mutations in TBX5 cause Holt-Oram syndrome, characterized by congenital heart defects and upper limb abnormalities

• It maintains atrial identity in post-natal cardiomyocytes by binding to and preserving tissue-specific chromatin architecture

What molecular weights should researchers expect when detecting TBX5 protein?

When using TBX5 antibodies for Western blot analysis, researchers should be aware of multiple isoforms with different molecular weights:

The detection of multiple bands may reflect post-translational modifications, as bioinformatic analysis of TBX5 indicates numerous potential phosphorylation sites and two potential sumoylation sites .

How can researchers distinguish between TBX5 isoforms using antibodies?

Distinguishing between TBX5 isoforms requires careful consideration of antibody epitope selection and experimental design:

• Epitope-specific antibodies: Use antibodies targeting the C-terminal region (absent in TBX5b) to specifically detect full-length TBX5a. Alternatively, use antibodies recognizing the N-terminal region to detect both isoforms .

• Subcellular fractionation: TBX5a localizes exclusively to the nucleus, while TBX5b is found in both nucleus and cytoplasm. Fractionation before Western blotting can help distinguish the isoforms based on their differential localization .

• Isoform-specific functional assays: The two isoforms have distinct biochemical properties. TBX5a functions as a transcriptional activator, while TBX5b may act as an antagonist. Transcriptional reporter assays can help distinguish their activities .

• Resolution optimization: Use gradient gels (e.g., 4-12%) to better separate the isoforms based on their significant molecular weight differences (35 kDa vs. 64-80 kDa) .

A specific example from research shows that using an antibody targeting the first 60 amino acids of murine Tbx5 detected both isoforms: TBX5a (64-80 kDa) in the 300 mM KCl nuclear fraction and multiple TBX5a-immunoreactive bands (64-80 kDa) in the 700 mM fraction, representing post-translationally modified forms .

How do TBX5 antibodies help elucidate the molecular mechanisms of Holt-Oram syndrome?

TBX5 antibodies have been instrumental in characterizing the functional defects caused by TBX5 missense mutations associated with Holt-Oram syndrome:

• DNA binding activity: Mutations G80R, R237Q, and R237W dramatically reduce TBX5's DNA-binding activity, as demonstrated through mobility shift assays with TBX5 antibodies .

• Transcriptional activation: TBX5 antibodies have revealed that mutations can affect transcriptional activation of target genes like ANF. Different mutations show varying effects, from complete loss to moderate reduction in activity .

• Protein-protein interactions: Immunoprecipitation with TBX5 antibodies demonstrated that all seven missense mutations studied (Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I) greatly reduced the interaction between TBX5 and NKX2.5 both in vivo and in vitro .

• Subcellular localization: Immunofluorescent staining with TBX5 antibodies showed that while wild-type TBX5 localizes exclusively to the nucleus, mutant proteins are found in both nucleus and cytoplasm, suggesting impaired nuclear localization .

These findings reveal that Holt-Oram syndrome results from multiple molecular mechanisms, depending on the specific mutation, which explains the phenotypic variability observed in patients.

How can TBX5 antibodies be used to study chromatin architecture and enhancer networks?

Recent research has employed TBX5 antibodies to understand how this transcription factor maintains atrial identity in post-natal cardiomyocytes through regulation of chromatin architecture:

• ChIP-seq applications: TBX5 antibodies can be used for chromatin immunoprecipitation followed by sequencing (ChIP-seq) to map genome-wide TBX5 binding sites. Research has shown that 69% of atrial-specific accessible chromatin regions are bound by TBX5 .

• Integration with ATAC-seq: Combining TBX5 ChIP-seq with ATAC-seq (Assay for Transposase-Accessible Chromatin) can reveal how TBX5 binding affects chromatin accessibility. TBX5 knockout studies showed downregulation of genes associated with TBX5-bound enhancers .

• Chromatin looping studies: TBX5 antibodies can be used in H3K27ac HiChIP experiments to identify TBX5-dependent chromatin loops. Research identified 510 chromatin loops sensitive to TBX5 dosage, with 74.8% of control-enriched loops containing anchors in control-enriched ATAC regions .

• Single-cell applications: TBX5 antibodies can be combined with single-nucleus RNA-seq and ATAC-seq to understand cell-type-specific roles of TBX5. This approach revealed that atrial cardiomyocytes from control and TBX5 knockout samples clustered separately, indicating profound transcriptional changes .

What are the optimal conditions for Western blot analysis of TBX5?

Based on published methodologies, the following protocol optimizations are recommended for TBX5 Western blot analysis:

• Sample preparation:

  • For tissue samples: Use RIPA buffer with protease inhibitors

  • Heart tissues require careful homogenization due to high connective tissue content

  • Nuclear fractionation may improve detection of nuclear-localized TBX5a

• Gel electrophoresis conditions:

  • Use reducing conditions with β-mercaptoethanol or DTT

  • 8-10% polyacrylamide gels for better resolution of high molecular weight TBX5 isoforms

  • Load 0.2 mg/mL of heart tissue lysate for optimal detection

• Membrane and blocking:

  • PVDF membrane shows better results than nitrocellulose for TBX5 detection

  • Block with 5% non-fat milk or BSA in TBS-Tween

• Antibody concentrations and incubation:

  • Primary antibody: 1-50 μg/mL depending on the specific antibody

  • For R&D Systems AF5918: Use at 1 μg/mL

  • For custom anti-Tbx5 antibody (first 60 aa): Use at 1:250 dilution

  • Secondary antibody: HRP-conjugated at 1:1000-1:5000 dilution

• Detection:

  • Enhanced chemiluminescence (ECL) detection system

  • Expect bands at 35 kDa (TBX5b) and/or 60-80 kDa (TBX5a and modified forms)

• Troubleshooting:

  • Non-specific bands may appear with some antibodies, particularly at 230 kDa with the Simple Western system

  • Multiple bands between 64-80 kDa likely represent post-translationally modified TBX5a

How should researchers validate TBX5 antibody specificity?

Proper validation of TBX5 antibody specificity is crucial for reliable results. Recommended validation approaches include:

• Positive and negative tissue controls:

  • Positive controls: Human/mouse heart tissue (high TBX5 expression)

  • Negative controls: Tissues known not to express TBX5

• Recombinant protein controls:

  • Test antibody against purified recombinant TBX5 protein

  • Example: E. coli-derived recombinant human TBX5 (Lys253-Lys327)

• Knockdown/knockout validation:

  • Compare TBX5 detection in wild-type versus TBX5 knockdown/knockout samples

  • Example: AAV9:Nppa-Cre-treated Tbx5Flox/Flox mice showed loss of TBX5 protein in atrial lysates

• Cross-reactivity testing:

  • Test against related T-box family members

  • The antibody described in did not recognize Tbx2, Tbx4, or Tbx20

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide to confirm signal specificity

• Overexpression validation:

  • Detect TBX5 in cells transfected with TBX5 expression vectors

  • Observe expected molecular weight shifts with tagged versions

• Isoform specificity:

  • Verify detection of both TBX5a (~64-80 kDa) and TBX5b (~35 kDa) isoforms

  • Confirm expected subcellular localization patterns (TBX5a: nuclear; TBX5b: nuclear and cytoplasmic)

What fixation and immunostaining protocols are optimal for TBX5 immunofluorescence?

For successful TBX5 immunofluorescence and immunocytochemistry, consider the following protocol optimizations:

• Fixation:

  • Paraformaldehyde fixation (2%) for 15-20 minutes at room temperature

  • Wash thoroughly with PBS after fixation

• Permeabilization:

  • Use 0.2% Tween in PBS for membrane permeabilization

  • Alternative: 0.1-0.5% Triton X-100 in PBS for 10 minutes

• Blocking:

  • Block with 5% BSA in PBS-0.2% Tween

  • Alternative: 3% nonfat milk in PBS for overnight primary antibody incubation

• Primary antibody incubation:

  • Anti-TBX5 antibody dilution: 1:250 to 1:1000 depending on the antibody

  • Incubate overnight at 4°C

• Secondary antibody:

  • Fluorescently labeled secondary antibodies (e.g., FITC-conjugated anti-rabbit or anti-sheep)

  • Dilution: 1:500 to 1:1000

  • Incubate for 1 hour at room temperature

• Nuclear counterstaining:

  • DAPI for nuclear visualization

  • Important for confirming nuclear localization of TBX5

• Mounting:

  • Use anti-fading mounting medium (e.g., Vectashield with DAPI)

• Imaging considerations:

  • Fluorescence microscopy with appropriate filter sets

  • Confocal microscopy for detailed subcellular localization studies

  • Z-stack imaging to confirm nuclear versus cytoplasmic distribution

• Expected results:

  • Wild-type TBX5a: Exclusively nuclear localization

  • TBX5b or mutant TBX5: Both nuclear and cytoplasmic distribution

How can researchers address common challenges in TBX5 antibody applications?

How should researchers interpret differences in TBX5 molecular weight across different studies?

Researchers should consider multiple factors when interpreting TBX5 molecular weight variations:

• Multiple isoforms: TBX5a (~64-80 kDa) and TBX5b (~35 kDa) result from alternative splicing

• Post-translational modifications: TBX5 contains numerous potential phosphorylation sites and two potential sumoylation sites, explaining the multiple bands between 64-80 kDa observed in some studies

• Species differences: Human and mouse TBX5 share 95% amino acid identity over residues 253-327, but differences exist in other regions

• Detection systems: Different molecular weights may be observed between traditional Western blot (~60-65 kDa) and Simple Western systems (~56 kDa) as seen with the R&D Systems antibody

• Experimental conditions: Reducing versus non-reducing conditions, different gel percentages, and buffer systems can affect apparent molecular weight

Understanding these factors is critical for proper data interpretation and experimental design in TBX5 research.

What emerging technologies could enhance TBX5 antibody utility in developmental biology research?

• Single-cell protein analysis: Adapting TBX5 antibodies for CyTOF or CODEX technology would enable simultaneous detection of TBX5 and other proteins at single-cell resolution during cardiac development

• In vivo antibody imaging: Development of fluorescently tagged anti-TBX5 antibody fragments (Fabs) for live imaging of TBX5 dynamics in developing embryos

• Proximity labeling approaches: TBX5 antibodies coupled with BioID or APEX2 systems to identify novel protein interaction partners in specific cellular compartments

• Super-resolution microscopy: Applying techniques like STORM or PALM with TBX5 antibodies to visualize the precise spatial organization of TBX5 within the nucleus

• Multiplexed chromatin studies: Combining TBX5 ChIP with other transcription factors and chromatin marks to build comprehensive models of cardiac enhancer function

These approaches could significantly advance our understanding of TBX5's role in cardiac development and provide new insights into congenital heart disease mechanisms.