TBX1 Antibody, Biotin conjugated

What is TBX1 and why is it significant in research?

TBX1 is a T-box transcription factor that plays a critical role in cardiovascular development by promoting pharyngeal arch segmentation during embryonic development. It is also involved in craniofacial muscle development and functions as a regulator of asymmetric cardiac morphogenesis by promoting expression of PITX2 alongside NKX2-5. TBX1 is required for the formation of thymus and parathyroid glands from the third pharyngeal pouch and contributes to hair follicle stem cell self-renewal . The gene has significant clinical relevance as haploinsufficiency of TBX1 is responsible for most physical malformations in DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS), characterized by hypoplastic thymus and parathyroid glands, congenital conotruncal cardiopathy, and characteristic facial dysmorphology . These disease associations make TBX1 a crucial target for developmental biology, immunology, and genetic disorder research.

What are the primary applications of TBX1 Antibody, Biotin conjugated?

TBX1 Antibody, Biotin conjugated products are primarily used in several key laboratory applications that leverage the biotin conjugation for enhanced detection sensitivity. The primary applications include:

Researchers should note that optimal working dilutions should be experimentally determined for each specific application and sample type . The biotin conjugation provides signal amplification capability when used with streptavidin detection systems, allowing for enhanced sensitivity in detecting low abundance TBX1 protein in complex biological samples.

What species reactivity can be expected with TBX1 Antibody, Biotin conjugated?

The species reactivity of commercially available TBX1 Antibody, Biotin conjugated products varies based on the antibody clone and manufacturing process. Based on available products:

When working with species not listed in confirmed reactivity, researchers should perform validation experiments before proceeding with full-scale studies. The cross-reactivity prediction is based on sequence homology analyses, but experimental confirmation is always recommended for critical research applications .

How should researchers optimize Western Blot protocols when using TBX1 Antibody, Biotin conjugated?

When optimizing Western Blot protocols with TBX1 Antibody, Biotin conjugated, researchers should focus on several critical parameters to ensure specific signal detection while minimizing background:

The recommended dilution range is 1:100-1:1000, but specific optimization should be performed for each new lot of antibody . For optimal results, researchers should prepare protein samples with complete protease inhibitor cocktails and use freshly prepared samples when possible. Since TBX1 is a nuclear protein (subcellular location: nucleus), appropriate nuclear extraction protocols should be employed to ensure efficient protein recovery .

For detection, utilize streptavidin-HRP secondary detection systems at appropriate dilutions (typically 1:2000-1:5000). When analyzing Western blot results, verify that the detected band corresponds to the expected molecular weight of TBX1 (approximately 37 kDa, though this may vary with post-translational modifications and isoforms). Additionally, researchers should include positive controls (e.g., cell lines known to express TBX1) and negative controls (e.g., TBX1 knockout samples or immunodepleted samples) to validate specificity.

To reduce non-specific binding, consider pre-incubating membranes with avidin/biotin blocking solutions before applying the biotinylated antibody, especially when working with tissues rich in endogenous biotin.

What are the special considerations for immunohistochemistry with paraffin-embedded sections using TBX1 Antibody, Biotin conjugated?

When performing immunohistochemistry with paraffin-embedded sections using TBX1 Antibody, Biotin conjugated, researchers should follow these methodological approaches:

Antigen retrieval is critical for detecting TBX1 in FFPE tissues. Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is recommended, with optimization for specific tissue types. The recommended dilution range for IHC-P is 1:100-1:500, but this should be experimentally determined for each application .

When using mouse-derived TBX1 antibodies (like NBP2-74470B) on mouse tissues, implement Mouse-on-Mouse blocking reagents to reduce background signal. Products such as PK-2200-NB and MP-2400-NB can be used for this purpose . Researchers should also employ endogenous biotin blocking steps, as many tissues (especially liver, kidney, and brain) contain high levels of endogenous biotin that can cause false-positive signals.

To develop the signal, use streptavidin-conjugated detection systems (HRP, AP, or fluorescent labels). For chromogenic detection, DAB (3,3'-diaminobenzidine) is commonly used with HRP systems. Include appropriate controls, including isotype controls and tissue sections known to be positive or negative for TBX1 expression.

How can researchers troubleshoot high background in immunohistochemistry when using TBX1 Antibody, Biotin conjugated?

High background is a common challenge when using biotin-conjugated antibodies in immunohistochemistry. The following methodological approaches can help troubleshoot and reduce background:

For mouse-derived antibodies used on mouse tissues (like NBP2-74470B), implement specific Mouse-on-Mouse blocking systems to prevent non-specific binding of the primary antibody to endogenous mouse immunoglobulins in the tissue . This is particularly important for TBX1 detection in mouse developmental studies.

Implement a comprehensive endogenous biotin blocking protocol using avidin-biotin blocking kits before applying the TBX1 Antibody, Biotin conjugated. This is essential because many mammalian tissues contain endogenous biotin that can directly bind to the streptavidin detection reagents.

Optimize antibody concentration through titration experiments, as excessive antibody concentrations often lead to increased background. Starting with higher dilutions (1:500) and working toward more concentrated solutions may help identify the optimal signal-to-noise ratio .

Extend washing steps (using PBS with 0.05-0.1% Tween-20) between each incubation step, and consider adding background reducing agents such as 1-5% BSA, 1-5% normal serum from the species in which the secondary reagent was raised, or commercial background reducers to the antibody diluent.

What are the most effective experimental controls when using TBX1 Antibody, Biotin conjugated?

Implementing appropriate controls is essential for generating reliable and interpretable data with TBX1 Antibody, Biotin conjugated. The following control strategy should be considered:

Positive Controls:

• Cell lines or tissues with known TBX1 expression (e.g., embryonic cardiac tissue)

• Recombinant TBX1 protein (where applicable)

• Tissues from wild-type animals at developmental stages with known TBX1 expression patterns

Negative Controls:

• TBX1 knockout or knockdown samples

• Tissues where TBX1 is not expressed

• Primary antibody omission control (tissue treated with all reagents except the primary antibody)

• Isotype control (using a biotin-conjugated antibody of the same isotype but irrelevant specificity)

Absorption Controls:

• Pre-incubation of the TBX1 Antibody, Biotin conjugated with the immunizing peptide to confirm specificity

• This is particularly relevant for polyclonal antibodies like bs-8257R-Biotin

Researchers should note that for monoclonal antibodies like NBP2-74470B, which is reactive to mouse and derived from mouse host, special attention to Mouse-on-Mouse blocking is necessary for IHC experiments to reduce background signal .

How can TBX1 Antibody, Biotin conjugated be used to study DiGeorge syndrome and related pathologies?

TBX1 Antibody, Biotin conjugated serves as a valuable tool for investigating DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS), as TBX1 haploinsufficiency is responsible for most physical malformations in these conditions . The following methodological approaches are recommended:

For human patient samples, immunohistochemistry using TBX1 Antibody, Biotin conjugated can help visualize altered expression patterns in affected tissues. This can be performed on tissues from patients with confirmed 22q11.2 deletions (the chromosomal region containing TBX1). Comparing TBX1 expression levels in patient samples versus controls can provide insights into how reduced TBX1 expression correlates with specific phenotypic manifestations.

In mouse models of DiGeorge syndrome, TBX1 Antibody, Biotin conjugated can be used to track developmental abnormalities in the pharyngeal arch arteries, thymus, parathyroid glands, and cardiac outflow tract . Detailed immunohistochemical studies at various embryonic stages can help establish the temporal relationship between TBX1 expression changes and the emergence of DGS-like phenotypes.

Western blotting with TBX1 Antibody, Biotin conjugated can quantify differences in TBX1 protein levels between affected and unaffected samples, allowing researchers to correlate protein expression with phenotype severity. This approach is particularly useful for analyzing tissues from heterozygous TBX1 knockout models, which mimic the haploinsufficiency seen in human patients.

What are the key considerations when selecting between HRP and Biotin conjugated TBX1 antibodies?

When deciding between HRP-conjugated (like NBP2-74470H ) and Biotin-conjugated (like NBP2-74470B ) TBX1 antibodies, researchers should consider several methodological factors:

For tissues with high endogenous biotin (such as liver, kidney, brain), HRP-conjugated antibodies may be preferable to avoid false positives . Conversely, for applications requiring maximum sensitivity, the biotin-streptavidin amplification system provided by biotin-conjugated antibodies offers significant advantages .

How can researchers accurately quantify TBX1 expression using TBX1 Antibody, Biotin conjugated?

Accurate quantification of TBX1 expression using TBX1 Antibody, Biotin conjugated requires robust methodological approaches:

For Western blot quantification, researchers should use calibrated protein standards alongside samples to generate a standard curve. Densitometric analysis should be performed using validated software (e.g., ImageJ), with normalization to appropriate loading controls such as GAPDH, β-actin, or nuclear proteins like Lamin B1 (since TBX1 is a nuclear protein) . Multiple technical replicates (at least 3) and biological replicates should be performed to ensure statistical validity.

For immunohistochemical quantification, several approaches can be employed:

• H-score method: Calculate the product of staining intensity (0-3) and percentage of positive cells

• Automated image analysis using software that can distinguish positive nuclei (TBX1 is predominantly nuclear) from negative nuclei

• Quantitative measurement of staining intensity in regions of interest

When comparing TBX1 expression between different conditions or genotypes, appropriate statistical tests should be applied based on data distribution. For normally distributed data, t-tests or ANOVA are appropriate; for non-normally distributed data, non-parametric tests such as Mann-Whitney U or Kruskal-Wallis should be used.

How do different antibody clones and conjugates affect experimental outcomes when studying TBX1?

The selection of specific TBX1 antibody clones and conjugates can significantly impact experimental outcomes:

Polyclonal antibodies (like bs-8257R-Biotin ) recognize multiple epitopes on the TBX1 protein, potentially offering higher sensitivity but with greater batch-to-batch variation. They are generated against specific immunogen ranges (e.g., 165-270/398 amino acids for bs-8257R-Biotin) , which may affect epitope accessibility in certain experimental conditions.

Monoclonal antibodies (like NBP2-74470B, clone OTI1C2 ) recognize a single epitope, providing higher specificity but potentially lower sensitivity. They offer consistent performance across batches but may be more vulnerable to epitope masking due to protein modifications or conformational changes.

The immunogen source also matters significantly. Antibodies raised against full-length recombinant protein (like NBP2-74470B, which uses full-length recombinant protein of human TBX1 ) may recognize different epitopes compared to those raised against synthetic peptides (like bs-8257R-Biotin, which uses KLH conjugated synthetic peptide ).

Researchers should carefully evaluate whether their experimental questions require broader epitope recognition (favoring polyclonal antibodies) or precise targeting of specific protein regions (favoring monoclonal antibodies). For studies involving protein modifications or interactions that might mask specific epitopes, using multiple antibodies recognizing different regions of TBX1 can provide complementary data.

How can TBX1 Antibody, Biotin conjugated be integrated into multiplexed staining protocols?

Multiplexed staining allows simultaneous detection of multiple targets in the same sample, providing valuable co-localization information. When incorporating TBX1 Antibody, Biotin conjugated into multiplexed protocols:

For fluorescence-based multiplexing, TBX1 Antibody, Biotin conjugated can be detected using streptavidin conjugated to fluorophores with spectral properties distinct from other detection systems in the protocol. Common fluorophores include Alexa Fluor 488, 555, 594, 647, or quantum dots. When designing panels, consider the nuclear localization of TBX1 and select membrane or cytoplasmic markers with distinct subcellular localization for clear signal separation.

For chromogenic multiplexing, researchers can use streptavidin-HRP followed by distinct chromogens (e.g., DAB for brown, Vector Red for red, Vector Blue for blue). Sequential staining protocols with appropriate blocking steps between each primary antibody are recommended to prevent cross-reactivity.

When combining TBX1 detection with other T-box family members, careful validation is essential due to potential cross-reactivity. The T-box DNA binding domain is highly conserved across family members, so antibodies targeting this region may cross-react with related proteins .

For co-localization studies with potential TBX1 interaction partners (such as NKX2-5 mentioned in the background information ), specialized proximity ligation assays can be employed using the biotin conjugation as one of the detection methods.

What approaches are recommended for studying TBX1 in developmental biology research?

TBX1's critical role in embryonic development makes it an important target for developmental biology research. The following methodological approaches are recommended:

For temporal expression studies, collect tissues at defined developmental stages and perform either Western blotting or immunohistochemistry with TBX1 Antibody, Biotin conjugated. This allows tracking of TBX1 expression changes throughout development, particularly in structures known to be affected in DiGeorge syndrome, such as the pharyngeal arches, thymus, and cardiac outflow tract .

For spatial expression mapping, whole-mount immunohistochemistry or serial section analysis can be performed with TBX1 Antibody, Biotin conjugated. This helps establish the precise tissue distribution of TBX1 protein at critical developmental timepoints. The biotin conjugation offers signal amplification advantages that can be particularly helpful for detecting low-level expression in early developmental stages.

Co-expression studies combining TBX1 Antibody, Biotin conjugated with antibodies against developmental markers (such as SOX9, PAX3, or FOXC2) can help establish the relationship between TBX1 expression and specific developmental processes. These studies should employ appropriate multiple-labeling techniques with careful consideration of antibody compatibility.

For functional studies, TBX1 expression can be compared between wild-type and genetically modified organisms (conditional knockouts, knockdowns, or mutations) to correlate protein expression changes with developmental phenotypes. The nuclear localization of TBX1 should be considered when designing studies examining its transcriptional regulatory functions.