TBX6 Antibody, HRP conjugated

What is TBX6 and why is it important in developmental biology research?

TBX6 (T-box transcription factor 6) is a critical transcription factor belonging to the T-box family that plays essential roles in embryonic development, particularly in mesoderm formation and somitogenesis. The protein contains a highly conserved DNA-binding domain called the T-box that recognizes specific DNA sequences to regulate gene expression during development. TBX6 is particularly significant in developmental biology research because it functions as a key regulator in the presomitic mesoderm (PSM), which gives rise to the vertebral column and associated musculature. The human TBX6 protein is identified by UniProt accession O95947 and is encoded by the TBX6 gene (GeneID: 6911) . Researchers frequently study TBX6 to understand developmental disorders affecting the axial skeleton and to elucidate fundamental mechanisms of embryonic patterning. Studying TBX6 provides insights into congenital vertebral malformations and other developmental disorders associated with mutations in this gene.

What are the key applications for TBX6 Antibody, HRP conjugated in research settings?

TBX6 Antibody, HRP conjugated serves multiple research applications in developmental biology and molecular cell research. The primary application documented in the product specifications is ELISA (Enzyme-Linked Immunosorbent Assay) . This antibody enables researchers to detect and quantify TBX6 protein levels in various experimental setups, particularly useful when investigating embryonic development processes or analyzing genetic developmental disorders. The horseradish peroxidase (HRP) conjugation provides direct enzymatic detection without requiring secondary antibodies, streamlining experimental workflows and potentially improving sensitivity when optimized properly. While the documented application is ELISA, researchers should note that polyclonal antibodies against TBX6 have been used in other experimental contexts in the literature, including immunoprecipitation studies investigating protein-protein interactions such as those between TBX6 and Ripply2 .

What protein regions does the TBX6 Antibody, HRP conjugated recognize?

The TBX6 Antibody, HRP conjugated is generated using a specific immunogen consisting of recombinant Human T-box transcription factor TBX6 protein, specifically amino acids 295-436 . This region encompasses the C-terminal portion of the protein, which contains important functional domains. Researchers should be aware that this antibody may not recognize all forms of TBX6, particularly truncated variants or those with specific mutations in the C-terminal region. For instance, in experimental settings studying Tbx6-venus fusion proteins, certain antibodies produced against the Tbx6 C-terminal peptide did not recognize the Tbx6-venus fusion protein . This information is crucial when designing experiments, as it determines whether the antibody will detect your protein of interest, especially when working with fusion proteins or truncated variants.

How should I optimize ELISA protocols using TBX6 Antibody, HRP conjugated?

When optimizing ELISA protocols with TBX6 Antibody, HRP conjugated, several critical parameters require careful consideration. Begin with antibody titration experiments to determine the optimal concentration for your specific sample type. While manufacturer specifications do not provide explicit dilution recommendations (stating "optimal dilutions/concentrations should be determined by the end user") , a good starting range is typically 1:500 to 1:2000 for HRP-conjugated antibodies in ELISA. The buffer composition (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) should inform your blocking and washing buffer selection to maintain antibody stability and function. Consider using phosphate buffers at similar pH for washing steps and develop the assay using appropriate HRP substrates like TMB (3,3',5,5'-Tetramethylbenzidine) with optimization of development time. Include both positive controls (recombinant TBX6 protein) and negative controls (samples known not to express TBX6) to validate assay specificity. Additionally, determine the linear range of detection for your experimental system and ensure your samples fall within this range, diluting as necessary.

What experimental considerations are important when studying TBX6 degradation mechanisms?

Research has revealed complex mechanisms regulating TBX6 protein stability, particularly through interaction with Ripply2. When designing experiments to study TBX6 degradation, several critical factors should be considered. First, the T-box domain, especially amino acids 125-152, plays a central role in the degradation of TBX6 protein in vivo . Experiments have shown that deletion of this region (Tbx6 Δ124-152aa) leads to stabilization and anterior expansion of TBX6 expression patterns in embryos . Second, while this region is important for degradation, it is not the only Ripply2-binding motif in the T-box. Co-immunoprecipitation assays have demonstrated that deletion of amino acids 125-152 does not affect the ability of TBX6 to form a complex with Ripply2 . This suggests that the mechanism of TBX6 degradation involves more than simple protein-protein interaction with Ripply2. When studying this phenomenon, researchers should employ multiple complementary approaches including both in vivo models (such as transgenic mice or chimeric embryos) and cell culture systems with inducible expression of wild-type and mutant TBX6 variants.

How can I validate the specificity of TBX6 Antibody, HRP conjugated in my experimental system?

Validating antibody specificity is crucial for obtaining reliable experimental results. For TBX6 Antibody, HRP conjugated, multiple validation strategies should be employed. First, perform positive and negative control experiments using samples with known TBX6 expression status. For positive controls, consider using cell lines or tissues known to express TBX6, such as differentiated embryonic stem cells treated with CHIR99021 (GSK inhibitor) which has been shown to induce TBX6 expression . For negative controls, use cell lines or tissues that do not express TBX6, or CRISPR/Cas9-generated TBX6 knockout cells. Second, conduct peptide competition assays using the immunogen peptide (recombinant Human TBX6 protein, amino acids 295-436) to demonstrate signal specificity. Third, consider orthogonal validation using alternative detection methods such as RT-PCR or RNA-seq to correlate protein detection with mRNA expression levels. For advanced validation, comparing detection patterns between different antibodies targeting distinct epitopes of TBX6 can provide additional confidence in specificity. Remember that this antibody has been documented to react with human TBX6 , so species cross-reactivity should be verified if working with models from other species.

How can TBX6 Antibody, HRP conjugated be utilized in studying embryonic stem cell differentiation?

TBX6 Antibody, HRP conjugated offers significant value in monitoring presomitic mesoderm (PSM) differentiation in embryonic stem (ES) cell models. Research protocols have demonstrated that dissociated feeder-free ES cells cultured in differentiation medium for 2 days, followed by treatment with 3 μM CHIR99021 (GSK inhibitor) for 2-4 days, efficiently induces TBX6 expression . In such experimental setups, the TBX6 Antibody, HRP conjugated can be employed in ELISA-based assays to quantitatively monitor the temporal dynamics of TBX6 expression during differentiation. This provides a precise measurement of differentiation efficiency and timing. The antibody can be particularly valuable when establishing new differentiation protocols, comparing efficiency between protocols, or assessing the impact of specific signaling modulators on mesoderm specification. By measuring TBX6 protein levels at multiple time points (day 2, 3, and 4 of differentiation), researchers can identify the optimal time window for peak TBX6 expression, which has been observed to typically peak at day 3 in CHIR99021-treated ES cells . This information is crucial for experiments requiring precise timing of cellular events during differentiation.

What insights can be gained from studying TBX6-Ripply2 interactions in developmental contexts?

The interaction between TBX6 and Ripply2 represents a sophisticated regulatory mechanism controlling vertebrate segmentation during embryonic development. Research has revealed that Ripply2 directly binds to TBX6 and mediates its degradation, establishing a critical boundary in the presomitic mesoderm . When investigating this interaction, researchers can gain several key insights: First, the precise spatial regulation of TBX6 through Ripply2-mediated degradation creates sharp expression boundaries essential for proper somite formation. Second, the T-box domain of TBX6, particularly amino acids 125-152, contains a degradation motif that is crucial for this process in vivo, though interestingly, deletion of this region does not prevent Ripply2 binding . This suggests a more complex mechanism than simple binding-induced degradation. Third, the ubiquitin-proteasome system likely plays a role in this degradation, as indicated by experimental approaches using ubiquitin detection systems . To study these interactions, researchers can employ co-immunoprecipitation assays, GST-pull-down experiments, and transgenic models expressing mutant forms of TBX6. Additionally, inducible expression systems in ES cells provide controlled environments to study the kinetics and requirements of TBX6 degradation.

How can CRISPR/Cas9-mediated approaches be utilized for studying TBX6 function?

CRISPR/Cas9 technology offers powerful approaches for studying TBX6 function in developmental contexts. Several sophisticated strategies have been employed in the literature. One approach involves generating knock-in embryonic stem (ES) cell lines where fluorescent reporter genes like venus are fused to endogenous TBX6 . This enables real-time visualization of TBX6 expression and localization without disrupting the endogenous regulatory elements. For example, researchers have successfully generated Tbx6-venus fusion knock-in ES cells by designing sgRNAs targeting Tbx6-intron-1 and using a puromycin selection cassette . Another approach involves creating specific mutations or deletions within functional domains of TBX6 to study structure-function relationships. For instance, deletion of amino acids 124-152 in the T-box domain has revealed crucial roles in protein degradation . When implementing CRISPR/Cas9 strategies, researchers should design sgRNAs with high specificity (for example: 5'-caccGTGAGCGGTTGGATTGGCTC-3') , include appropriate selection markers for enriching edited cells, and validate genomic modifications through sequencing and functional assays. For complex manipulations like domain deletions or reporter fusions, designing precise homology-directed repair templates with homology arms of appropriate length (approximately 1kb) is critical for efficient integration.

What are common challenges when working with TBX6 Antibody, HRP conjugated and how can they be addressed?

Researchers working with TBX6 Antibody, HRP conjugated may encounter several technical challenges. One common issue is degradation of antibody performance over time due to improper storage. The manufacturer recommends storing the antibody at -20°C or -80°C and avoiding repeated freeze-thaw cycles . To mitigate this, prepare small aliquots upon receipt and thaw only what is needed for immediate use. Another challenge is nonspecific background signal in ELISA applications. This can be addressed by optimizing blocking buffers (consider 3-5% BSA or animal serum that matches the host species of the primary antibody) and including appropriate negative controls in each experiment. The high glycerol content (50%) in the antibody solution may affect pipetting accuracy; compensate by using reverse pipetting techniques and equilibrating the antibody to room temperature before pipetting. Additionally, if detection sensitivity is insufficient, consider extended substrate incubation times, ensuring the substrate is fresh, or employing signal amplification systems compatible with HRP. For high background in immunoassays, increasing wash frequency and duration, and optimizing antibody concentration through careful titration experiments can improve signal-to-noise ratios.

How can I troubleshoot inconsistent results when studying TBX6 protein stability?

Inconsistent results when studying TBX6 protein stability may stem from several sources. First, consider the temporal dynamics of TBX6 expression, which has been shown to have specific patterns during differentiation, with expression typically peaking at day 3 in CHIR99021-treated ES cells . Ensuring consistent timing for sample collection is crucial. Second, the stability of TBX6 is influenced by specific protein domains, particularly the T-box region containing amino acids 125-152 . When using mutant or fusion proteins, verify that these constructs behave as expected through appropriate validation experiments. Third, the Ripply2-mediated degradation mechanism involves complex protein-protein interactions that may be sensitive to experimental conditions . Standardize buffer compositions, incubation times, and temperatures across experiments. Fourth, inconsistencies may arise from variability in proteasome activity between experimental batches. Consider including proteasome inhibitors as controls to distinguish between proteasome-dependent and independent mechanisms. Finally, when using inducible expression systems, verify consistent expression levels between experiments using techniques like qRT-PCR or western blotting. For chimeric embryo experiments, account for variation in contribution of mutant cells to tissues by using appropriate internal controls and analyzing sufficient numbers of embryos to account for biological variability.

What controls are essential when working with TBX6 Antibody, HRP conjugated?

Implementing rigorous controls is essential for generating reliable data with TBX6 Antibody, HRP conjugated. For ELISA applications, include the following controls: First, technical negative controls where the primary antibody is omitted but all other reagents are included to assess non-specific binding of detection components. Second, biological negative controls using samples known not to express TBX6, such as undifferentiated ES cells or tissues where TBX6 is not expressed . Third, positive controls using recombinant TBX6 protein or samples with confirmed TBX6 expression, such as day 3 CHIR99021-treated ES cells . Fourth, standard curves using purified recombinant TBX6 protein at known concentrations to enable quantification. For experiments studying TBX6 degradation or protein interactions, additional controls are necessary: include wild-type TBX6 alongside mutant variants (such as Tbx6 Δ124-152aa) to compare stability differences ; use co-immunoprecipitation with non-relevant proteins to control for non-specific binding; and employ multiple detection methods (anti-GFP for fusion proteins and anti-TBX6 for endogenous protein) to distinguish between different protein forms when working with fusion constructs . For all experimental approaches, biological replicates are essential to account for variability.

What emerging techniques show promise for studying TBX6 protein interactions and function?

Several emerging techniques offer new possibilities for studying TBX6 biology with increased precision. Proximity labeling approaches, such as BioID or TurboID, could provide insights into the dynamic TBX6 interactome in living cells by identifying proteins that transiently interact with TBX6 during development. Single-cell proteomics techniques may reveal cell-to-cell variability in TBX6 protein levels and post-translational modifications during differentiation, offering new insights into the heterogeneity of developmental processes. Advanced imaging techniques, including super-resolution microscopy combined with live cell imaging of fluorescent TBX6 fusion proteins (similar to the Tbx6-venus constructs described in the literature ), could reveal the real-time dynamics of TBX6 degradation at the single-molecule level. CRISPR activation and interference (CRISPRa/CRISPRi) systems targeting TBX6 regulatory elements could help map the gene regulatory networks controlled by TBX6 during development. Finally, organoid models of somitogenesis, combined with the TBX6 Antibody, HRP conjugated for protein detection, may provide more physiologically relevant contexts for studying TBX6 function compared to conventional 2D cell culture systems, bridging the gap between in vitro and in vivo studies.

How might TBX6 research contribute to understanding congenital vertebral malformations?

TBX6 research has significant implications for understanding congenital vertebral malformations, as evidenced by its association with disorders affecting the axial skeleton (OMIM: 122600) . Future research directions could explore several key areas. First, detailed characterization of patient-derived TBX6 mutations using CRISPR/Cas9-engineered cell lines and mouse models could establish genotype-phenotype correlations, potentially enabling more precise prognosis for patients with specific TBX6 variants. Second, investigating how TBX6 degradation mechanisms, particularly the Ripply2-mediated pathway , contribute to vertebral patterning defects could identify molecular targets for therapeutic intervention. Third, exploring the interaction between TBX6 and other developmental pathways (such as Notch, Wnt, and FGF signaling) in the context of somitogenesis could reveal how these pathways integrate to ensure proper vertebral formation. Screening compounds that modulate TBX6 stability or function, perhaps by targeting the critical T-box domain or the interaction with Ripply2 , might lead to therapeutic approaches for preventing or ameliorating vertebral malformations during embryonic development. Additionally, population genetics studies examining TBX6 variants across different ethnic groups could identify risk factors and protective variants that influence susceptibility to vertebral malformations.

What are the limitations of current TBX6 antibodies and how might next-generation reagents overcome these challenges?

Current TBX6 antibodies, including the HRP-conjugated version, present several limitations that next-generation reagents could address. First, existing antibodies have limited documented applications, primarily ELISA , restricting their utility in diverse experimental contexts. Future antibodies with validated applications across multiple techniques (immunohistochemistry, ChIP-seq, flow cytometry) would greatly expand research possibilities. Second, current antibodies may have epitope accessibility issues in certain experimental conditions or when TBX6 is in complex with other proteins like Ripply2 . Next-generation antibodies targeting different epitopes or conformation-specific antibodies could overcome these limitations. Third, the polyclonal nature of current antibodies introduces batch-to-batch variability. Developing monoclonal antibodies or recombinant antibody fragments with defined epitope recognition would enhance reproducibility. Fourth, species cross-reactivity is limited with current antibodies primarily targeting human TBX6 . Antibodies recognizing conserved epitopes across multiple species would facilitate comparative studies. Finally, advanced reagents such as nanobodies (single-domain antibodies) conjugated to bright fluorophores, split protein complementation tags for studying protein-protein interactions, or proximity-dependent labeling enzymes fused to anti-TBX6 antibodies would enable new experimental approaches for studying TBX6 biology in living systems with minimal perturbation of function.

What are the optimal storage and handling conditions for TBX6 Antibody, HRP conjugated?

Proper storage and handling of TBX6 Antibody, HRP conjugated is essential to maintain its activity and specificity. According to manufacturer specifications, the antibody should be stored at -20°C or -80°C immediately upon receipt . The liquid formulation contains 50% glycerol, which prevents freezing at -20°C and helps maintain antibody stability during storage. To minimize activity loss from repeated freeze-thaw cycles, it is recommended to prepare small working aliquots upon receipt and thaw only what is needed for immediate use . When handling the antibody, allow it to equilibrate to room temperature before opening to prevent condensation, which can introduce contaminants and accelerate degradation. The buffer composition (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, PH 7.4) is designed to maintain antibody stability, but exposure to light should be avoided, particularly due to the HRP conjugation, as photobleaching can reduce enzymatic activity . During experimental procedures, keep the antibody on ice when not in use, and return to storage promptly after completing the protocol. For long-term storage beyond one year, -80°C is preferable to -20°C to better preserve activity. Following these guidelines will ensure maximum sensitivity and specificity in your experiments.

What is the detailed purification method for TBX6 Antibody, HRP conjugated, and how does it affect experimental applications?

TBX6 Antibody, HRP conjugated undergoes purification using Protein G, achieving a purity level exceeding 95% . This purification method has several important implications for experimental applications. Protein G is an immunoglobulin-binding protein with high affinity for the Fc region of IgG antibodies, particularly from rabbit sources (the host species for this antibody) . The purification process effectively removes contaminating proteins, non-specific antibodies, and serum components that could contribute to background signal in sensitive applications like ELISA. The high purity level (>95%) ensures consistent performance across experiments and reduces lot-to-lot variability. The purification method preserves the antigen-binding capacity while maintaining the structural integrity of both the antibody and the conjugated HRP enzyme. This is particularly important for ensuring optimal enzymatic activity in detection applications. Researchers should be aware that Protein G purification primarily selects for IgG antibodies, and the polyclonal nature of this antibody means that it contains a mixture of antibodies recognizing different epitopes within the immunogen region (recombinant Human TBX6 protein, amino acids 295-436). For applications requiring extreme specificity to a single epitope, monoclonal antibodies might be preferable, though they would not offer the epitope diversity that can be advantageous in certain detection scenarios.

What are the detailed properties of the TBX6 protein that this antibody detects?

The TBX6 Antibody, HRP conjugated recognizes T-box transcription factor TBX6, a protein with several notable properties important for researchers to consider. Human TBX6 is identified by UniProt Primary Accession O95947 and is also known by secondary accessions Q8TAS4 and Q9HA44 . The gene encoding TBX6 (gene symbol: TBX6, GeneID: 6911) is associated with congenital vertebral malformations in the OMIM database (entry 122600) . The TBX6 protein belongs to the T-box family of transcription factors, characterized by a highly conserved DNA-binding domain called the T-box. The immunogen used to generate this antibody corresponds to amino acids 295-436 of the human TBX6 protein , a region in the C-terminal portion that contains functional domains important for protein-protein interactions. Research has shown that specific regions of the TBX6 protein, particularly amino acids 125-152 within the T-box domain, play crucial roles in protein degradation pathways mediated by Ripply2 . This region appears to contain a degradation motif essential for proper embryonic patterning. Interestingly, while this region is important for degradation, experimental evidence indicates that it is not the only Ripply2-binding motif in the T-box, suggesting complex regulatory mechanisms controlling TBX6 stability and function .

How should researchers choose between different commercial sources of TBX6 antibodies?

When selecting between commercial sources of TBX6 antibodies, researchers should evaluate several critical factors to ensure optimal performance in their specific applications. First, compare the immunogen used to generate the antibodies. Both AFG Scientific and Abbexa offer TBX6 antibodies generated against the same immunogen (recombinant Human T-box transcription factor TBX6 protein, amino acids 295-436) , suggesting similar epitope recognition profiles. Second, review the validated applications for each antibody. Both vendors specify ELISA as the validated application , but if other applications are critical for your research, additional validation would be necessary regardless of vendor. Third, examine technical specifications such as purity level (both >95%) , host species (both rabbit) , and buffer composition (both contain 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) . Fourth, consider customer support and validation data availability—vendors providing detailed application notes, representative data, and responsive technical support may offer advantages despite similar product specifications. Fifth, assess cost-effectiveness by comparing not just price but also concentration and amount provided. Finally, consider peer recommendations and citations in relevant literature, as successful use in published studies similar to your research provides confidence in performance. If possible, request small-scale testing samples from multiple vendors to directly compare performance in your specific experimental system before committing to larger purchases.

How might advances in antibody technology impact future TBX6 research?

Advances in antibody technology are poised to significantly enhance TBX6 research capabilities in several key areas. Recombinant antibody production techniques will likely lead to development of renewable TBX6 antibodies with precisely defined properties, eliminating the batch-to-batch variability inherent to conventional polyclonal antibodies like the current HRP-conjugated TBX6 antibody . This will enable more reproducible long-term studies of TBX6 in developmental contexts. Single-domain antibodies (nanobodies) derived from camelid immunoglobulins offer smaller size and superior tissue penetration, potentially allowing better access to TBX6 in complex tissues or protein complexes where conventional antibodies may have limited access. Site-specific conjugation technologies will enable precise control over the position and number of reporter molecules (like HRP) attached to TBX6 antibodies, optimizing signal-to-noise ratios and detection sensitivity. Bispecific antibodies simultaneously targeting TBX6 and interaction partners like Ripply2 could enable direct visualization of protein complexes in situ, providing unprecedented insights into the spatial organization of these interactions during development. Finally, intrabodies—antibodies engineered to function within living cells—may eventually allow real-time tracking of endogenous TBX6 without the need for genetic manipulation through fusion proteins. These technological advances will collectively expand the experimental toolkit available to researchers studying TBX6's role in development and disease, enabling more sophisticated investigations of this critical developmental regulator.

What interdisciplinary approaches might advance our understanding of TBX6 function in development and disease?

Understanding TBX6 function in development and disease will increasingly require interdisciplinary approaches that integrate multiple technologies and perspectives. Computational biology approaches, including protein structure prediction and molecular dynamics simulations, could provide insights into how specific domains of TBX6, such as the critical region between amino acids 125-152 , contribute to protein stability and interaction with partners like Ripply2. Systems biology approaches integrating transcriptomics, proteomics, and epigenomics data could map the complete gene regulatory networks downstream of TBX6, revealing its broader developmental impact. Patient-derived induced pluripotent stem cells (iPSCs) differentiated along the mesoderm lineage while monitoring TBX6 expression using antibody-based methods could create personalized models of vertebral malformations associated with TBX6 mutations. Bioengineering approaches, such as microfluidic devices mimicking the physical and chemical gradients present during somitogenesis, could provide controlled environments for studying TBX6 dynamics under physiologically relevant conditions. Advanced imaging techniques combined with genome editing to create endogenously tagged TBX6 (similar to the Tbx6-venus system ) could enable real-time visualization of TBX6 behavior during development. Finally, collaborative efforts between developmental biologists, clinicians, and geneticists studying patients with vertebral malformations will be essential to translate basic research findings into clinical applications, potentially leading to early interventions for TBX6-associated developmental disorders.