TFAP2B Antibody, FITC conjugated

What is TFAP2B and what cellular functions does it regulate?

TFAP2B (Transcription Factor AP-2 beta, Activating Enhancer Binding Protein 2 Beta) is a sequence-specific DNA-binding protein that interacts with inducible viral and cellular enhancer elements to regulate transcription of selected genes. AP-2 factors bind to the consensus sequence 5'-GCCNNNGGC-3' and activate genes involved in a spectrum of important biological functions including proper eye, face, body wall, limb and neural tube development. They also suppress a number of genes including MCAM/MUC18, C/EBP alpha and MYC. AP-2-beta appears to be required for normal face and limb development and for proper terminal differentiation and function of renal tubular epithelia .

How does FITC conjugation enhance detection capabilities compared to unconjugated TFAP2B antibodies?

FITC (Fluorescein isothiocyanate) conjugation provides direct fluorescence visualization capabilities without requiring secondary antibody detection steps. This conjugation allows researchers to:

• Perform direct immunofluorescence detection with reduced background

• Utilize the antibody in multiple-labeling experiments

• Conduct flow cytometry analysis with high sensitivity

• Reduce experimental time by eliminating secondary antibody incubation steps

The FITC-conjugated TFAP2B antibody emits green fluorescence (peak emission ~520nm) when excited at the appropriate wavelength (~495nm), allowing for direct visualization in fluorescence-based applications such as flow cytometry (FACS) without additional detection reagents .

What factors should be considered when selecting between polyclonal and monoclonal TFAP2B antibodies?

Selection should be based on the specific research application, with monoclonal antibodies preferred when absolute specificity is critical, and polyclonal antibodies when signal amplification is needed for low-abundance targets .

What are the optimal protocols for using TFAP2B antibody, FITC conjugated in immunofluorescence microscopy?

For optimal immunofluorescence microscopy using FITC-conjugated TFAP2B antibody, follow this protocol:

• Sample Preparation:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

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

• Antibody Incubation:

  • Dilute FITC-conjugated TFAP2B antibody (1:100 to 1:500 depending on signal strength required)

  • Incubate overnight at 4°C in a humidified chamber protected from light

  • Wash 3x with PBS containing 0.1% Tween-20

• Nuclear Counterstaining:

  • Counterstain with DAPI (1μg/ml) for 5 minutes

  • Mount with anti-fade mounting medium

• Controls:

  • Include an isotype control with FITC-conjugated IgG at the same concentration

  • Use a known TFAP2B-positive cell line as a positive control (e.g., MCF7)

Based on published protocols, researchers have successfully used TFAP2B antibodies at 1:100 dilution for neuroblastoma cell studies, with detection using fluorophore-conjugated secondary antibodies .

How should TFAP2B antibody concentration be optimized for different cell types?

Optimization of TFAP2B antibody concentration is critical for achieving specific staining without background across different cell types:

• Initial Titration:

  • Prepare a dilution series (1:50, 1:100, 1:200, 1:500, 1:1000)

  • Test on positive control cells known to express TFAP2B (e.g., neuroblastoma cell lines IMR-32, SH-EP)

  • Evaluate signal-to-noise ratio at each concentration

• Cell Type-Specific Considerations:

  • Neuroblastoma cells: Start with 1:100 dilution as used in published research

  • Lung cancer cells (e.g., H1299): May require higher antibody concentration (1:50-1:100) based on variable expression levels

  • MCF7 breast cancer cells: Effective at 1:1000 for Western blot, may need 1:200-1:500 for IF

• Expression Level Adjustment:

  • For cell types with high TFAP2B expression: Use more dilute antibody (1:500-1:1000)

  • For cell types with low TFAP2B expression: Use more concentrated antibody (1:50-1:200)

  • Confirm with Western blot to determine relative expression levels before IF optimization

The optimal antibody concentration balances specific signal detection while minimizing non-specific background fluorescence .

How can TFAP2B antibody, FITC conjugated be utilized to investigate transcriptional regulation mechanisms?

TFAP2B antibody, FITC conjugated can be leveraged to investigate transcriptional regulation through several advanced techniques:

• Chromatin Immunoprecipitation followed by Flow Cytometry (ChIP-Flow):

  • Use FITC-conjugated TFAP2B antibody to perform ChIP-Flow

  • This allows simultaneous analysis of TFAP2B binding to consensus sequence 5'-GCCNNNGGC-3' and other cellular parameters

  • Sort cells based on TFAP2B binding profiles for downstream analysis

• Co-localization with Other Transcription Factors:

  • Perform multi-color immunofluorescence combining FITC-conjugated TFAP2B antibody with differently labeled antibodies against other transcription factors

  • Assess co-localization at target gene promoters

  • Quantify Pearson's correlation coefficients between TFAP2B and factors like MYCN or REST, which are known to be regulated in neuroblastoma contexts

• Live Cell Imaging of Transcriptional Dynamics:

  • Use cell-permeable variants of FITC-conjugated TFAP2B antibodies for live cell imaging

  • Track transcription factor recruitment to enhancer elements in real-time

  • Correlate with gene expression changes measured by reporter constructs

Research has shown that TFAP2B regulates noradrenergic neuronal differentiation in neuroblastoma cells through modulation of DBH and TH gene expression, and down-regulation of MYCN and REST, demonstrating its critical role in transcriptional networks governing neuronal differentiation .

What role does TFAP2B play in cancer progression and how can FITC-conjugated antibodies help elucidate these mechanisms?

TFAP2B has significant implications in cancer progression that can be investigated using FITC-conjugated antibodies:

• Prognostic Value in Multiple Cancers:

  • In lung adenocarcinomas: Strong TFAP2B expression shows positive correlation with poor prognosis (P < 0.001)

  • In neuroblastoma: Low TFAP2B expression is associated with poor outcome and DNA methylation of the TFAP2B locus

• Signaling Pathway Analysis:

  • FITC-conjugated TFAP2B antibodies can be used in flow cytometry to analyze how TFAP2B affects key oncogenic pathways:

    • ERK/p38 signaling pathway

    • Caspase/cytochrome-c apoptotic pathway

    • VEGF/PEDF-dependent angiogenesis pathway

• Methylation Status Correlation:

  • Combine FITC-conjugated TFAP2B antibody staining with methylation-specific probes

  • Flow cytometric analysis can reveal inverse correlation between TFAP2B expression and CpG methylation of the TFAP2B locus

  • Research has demonstrated that demethylation with 5-aza-2′-deoxycytidine induces TFAP2B expression in vitro

• Therapeutic Target Potential:

  • FITC-conjugated TFAP2B antibodies can be used to screen compounds that modulate TFAP2B expression

  • Monitoring TFAP2B levels in response to treatment can help determine efficacy

  • Research indicates TFAP2B plays a critical role in regulating lung adenocarcinoma growth and could serve as a promising therapeutic target

How can researchers address non-specific binding when using TFAP2B antibody, FITC conjugated?

Non-specific binding is a common challenge when using FITC-conjugated antibodies. To minimize this issue with TFAP2B antibodies:

• Optimization of Blocking Conditions:

  • Increase blocking time (1-2 hours)

  • Test different blocking agents (5% BSA, 10% normal serum, commercial blocking buffers)

  • Add 0.1-0.3% Triton X-100 to blocking buffer for better penetration

• Antibody Dilution Optimization:

  • Perform a titration series (1:50 to 1:1000)

  • The optimal dilution balances specific signal without background

  • For polyclonal TFAP2B antibodies such as ABIN7172481, a higher dilution may reduce non-specific binding

• Washing Protocol Enhancement:

  • Increase number of washes (5-6 times instead of 3)

  • Use PBS-T (PBS with 0.1% Tween-20) for washing

  • Extend washing times to 10 minutes per wash

• Pre-absorption Controls:

  • Pre-incubate the FITC-conjugated TFAP2B antibody with recombinant TFAP2B protein

  • This competitive binding approach can confirm specificity

  • Use the immunogen peptide (e.g., AA 111-208 for ABIN7172481) for pre-absorption

• Appropriate Controls:

  • Include isotype controls (FITC-conjugated IgG of same species)

  • Use TFAP2B-negative cell lines as negative controls

  • Include positive controls (e.g., MCF7 cells which have confirmed TFAP2B expression)

These approaches will significantly improve signal-to-noise ratio in TFAP2B detection experiments.

How should researchers interpret contradictory results between TFAP2B protein detection and gene expression data?

When facing discrepancies between TFAP2B protein levels and gene expression data, consider these methodological approaches:

• Epigenetic Regulation Assessment:

  • Investigate DNA methylation status of TFAP2B promoter

  • Research has shown that low TFAP2B expression is strongly associated with CpG methylation of the TFAP2B locus in neuroblastomas

  • Consider treatment with demethylating agents (e.g., 5-aza-2′-deoxycytidine) to verify methylation-based silencing

• Post-transcriptional Regulation:

  • Examine microRNA-mediated regulation of TFAP2B mRNA

  • Assess mRNA stability through actinomycin D chase experiments

  • Investigate alternative splicing patterns that might affect antibody detection but not qPCR measurements

• Protein Degradation Pathways:

  • Investigate proteasomal degradation using inhibitors (MG132)

  • Assess protein half-life through cycloheximide chase assays

  • Examine post-translational modifications that might affect antibody epitope recognition

• Methodological Validation Steps:

  • Use multiple antibodies targeting different TFAP2B epitopes

  • Compare results across techniques (Western blot, immunofluorescence, flow cytometry)

  • Verify antibody specificity using TFAP2B knockdown or overexpression systems

• Quantitative Considerations:

  • Perform absolute quantification of both transcript and protein

  • Consider time-course experiments to identify temporal relationships

  • Establish threshold levels for biological significance

In published research, TFAP2B re-expression in neuroblastoma cells significantly impaired proliferation and cell cycle progression and induced neuronal differentiation, providing functional validation of TFAP2B activity beyond simple expression measurement .

How is TFAP2B antibody being utilized in neuroblastoma research?

TFAP2B antibody has become an important tool in neuroblastoma research, with several key applications:

• Prognostic Biomarker Assessment:

  • Flow cytometry using FITC-conjugated TFAP2B antibodies allows quantitative measurement of TFAP2B expression

  • Low TFAP2B expression is associated with poor outcome in primary neuroblastomas (studied in cohorts of up to 649 patients)

  • TFAP2B expression correlates with established prognostic factors through Wilcoxon rank-sum test analysis

• Differentiation Pathway Analysis:

  • Immunofluorescence staining with TFAP2B antibodies helps visualize cellular differentiation

  • TFAP2B induces neuronal differentiation in IMR-32 neuroblastoma cells

  • This is accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST

• Epigenetic Regulation Studies:

  • TFAP2B silencing through CpG methylation can be studied using combined approaches:

    • Methylation array analysis of the TFAP2B locus

    • TFAP2B protein detection by antibody-based methods

    • Re-expression studies following 5-aza-2′-deoxycytidine treatment

• Functional Studies using Gene Manipulation:

  • TFAP2B re-expression studies using tetracycline-inducible systems validate antibody specificity

  • shRNA-mediated TFAP2B knockdown experiments confirm antibody sensitivity

  • Antibody detection is crucial for confirming successful genetic manipulation

The combination of these approaches has revealed that TFAP2B mediates noradrenergic neuronal differentiation of neuroblastoma cells, suggesting potential therapeutic avenues for inducing differentiation in aggressive neuroblastomas .

What methodologies have proven effective for investigating TFAP2B's role in lung cancer progression?

TFAP2B has emerged as a critical factor in lung adenocarcinoma progression. Several methodological approaches using TFAP2B antibodies have yielded significant insights:

• Immunohistochemical Analysis of Clinical Samples:

  • TFAP2B antibody staining of tissue microarrays from lung adenocarcinoma patients

  • Envision+ Kit/HRP methodology with antigen retrieval (pH 9 buffer, 108°C for 15 min)

  • Correlation of TFAP2B expression with clinical outcomes showed strong TFAP2B expression has positive correlation with poor prognosis (P < 0.001)

• Apoptosis Assessment Following TFAP2B Modulation:

  • Flow cytometry with Annexin V-FITC/PI staining after TFAP2B siRNA transfection

  • H1299 lung cancer cells showed increased apoptosis following TFAP2B knockdown

  • This methodology revealed TFAP2B's role in regulating apoptotic pathways

• Signaling Pathway Analysis:

  • Western blot analysis of key signaling molecules following TFAP2B knockdown or overexpression

  • Revealed TFAP2B-mediated modulation of the ERK/p38, caspase/cytochrome-c, and VEGF/PEDF-dependent signaling pathways

  • These pathways were directly linked to cell growth, apoptosis, and angiogenesis in NSCLC cells

• In Vivo Xenograft Models:

  • Subcutaneous xenograft models using NSCLC cells with TFAP2B knockdown

  • Tumor growth monitoring and immunohistochemical analysis of harvested tumors

  • Confirmed TFAP2B's critical role in regulating lung adenocarcinoma growth in vivo

These complementary approaches have established TFAP2B as a promising therapeutic target for lung cancer treatment, with significant implications for personalized medicine approaches .

What emerging applications of TFAP2B antibody, FITC conjugated show promise for developmental biology research?

Several innovative applications of TFAP2B antibody, FITC conjugated are showing potential for advancing developmental biology research:

• Single-cell Analysis of Developmental Trajectories:

  • Flow cytometry with FITC-conjugated TFAP2B antibody enables sorting of specific cell populations

  • Combined with single-cell RNA sequencing, this approach can map TFAP2B's role in developmental lineage specification

  • Particularly relevant for studying face, limb, and neural tube development, where TFAP2B is known to be essential

• Organoid Development Monitoring:

  • Live imaging of TFAP2B expression in developing organoids

  • Correlation with morphological changes and differentiation markers

  • Potential for understanding renal tubular epithelia development where TFAP2B plays a functional role

• CRISPR-engineered Reporter Systems:

  • Creation of TFAP2B-GFP knock-in models to track endogenous expression

  • Validation using FITC-conjugated TFAP2B antibodies

  • Allows for dynamic tracking of TFAP2B expression during developmental processes

• Multiplex Imaging Technologies:

  • Incorporation of FITC-conjugated TFAP2B antibodies in multiplexed imaging platforms

  • Simultaneous visualization of TFAP2B with other developmental markers

  • Spatial mapping of transcription factor networks during embryogenesis

These emerging applications will help resolve the complex role of TFAP2B in orchestrating developmental processes, particularly in face, limb, and neural tube formation, as well as renal tubular epithelia differentiation .

How might advances in antibody technology enhance TFAP2B detection sensitivity and specificity?

Emerging technologies promise to enhance TFAP2B detection capabilities:

• Nanobody and Single-domain Antibody Approaches:

  • Development of FITC-conjugated TFAP2B nanobodies with superior tissue penetration

  • Smaller size (15kDa vs 150kDa) allows access to sterically hindered epitopes

  • Potential for higher density labeling and improved signal-to-noise ratio

• Proximity Ligation Assay (PLA) Integration:

  • Combining FITC-conjugated TFAP2B antibodies with PLA technology

  • Enables detection of protein-protein interactions involving TFAP2B

  • Offers exponential signal amplification for detection of low-abundance complexes

• Super-resolution Microscopy Optimization:

  • Development of TFAP2B antibodies with photoswitchable fluorophores

  • Enables STORM/PALM super-resolution imaging of TFAP2B localization

  • Potential to resolve nuclear distribution patterns at previously unattainable resolution

• Machine Learning-assisted Analysis:

  • AI algorithms to enhance signal detection and pattern recognition in TFAP2B staining

  • Automated quantification of nuclear localization and co-localization patterns

  • Reduction of subjective interpretation in complex tissue samples