TCF19 Antibody, Biotin conjugated

What is TCF19 and why is it significant for research?

TCF19 (Transcription Factor 19), also known as SC1, functions as a key epigenetic reader protein originally identified as a growth-regulated cDNA. TCF19 contains critical functional domains including a forkhead association (FHA) domain, a proline-rich region, and a plant homeodomain (PHD) or RING finger region at its carboxyl terminus. The FHA domain may serve as a nuclear signaling domain or a phosphoprotein binding domain, similar to well-known cell cycle proteins like Ki-67 and Chk2. The proline-rich region is characteristic of transactivating factors, while the PHD/RING finger region allows interaction with chromatin via methylated histone H3 .

TCF19 has significant research importance due to its:

• Association with type 1 diabetes susceptibility locus at chromosome 6p31.3

• Critical role in pancreatic β-cell proliferation and survival

• Involvement in cell cycle regulation and transcriptional control

• Emerging role in hepatocellular carcinoma and non-small cell lung carcinoma progression

• Regulation of glucose homeostasis and repression of de novo glucose production

What advantages does biotin conjugation offer for TCF19 antibodies?

Biotin conjugation provides several significant advantages for TCF19 antibody applications in research:

The biotin-streptavidin/avidin interaction is one of the strongest non-covalent biological bonds known, with remarkably high affinity and specificity. This property enables robust detection systems with exceptional stability across various experimental conditions. Multiple biotin molecules (>4) can be conjugated to each antibody molecule, which, when combined with the tetravalent binding mode of streptavidin, creates a powerful signal amplification system. This amplification enables detection of low-abundance targets like TCF19, which might otherwise be difficult to visualize with direct labeling methods .

Biotinylated TCF19 antibodies offer exceptional versatility across numerous research applications including immunohistochemistry (IHC), immunofluorescence (IF), immunocytochemistry (ICC), ELISA, western blotting, flow cytometry, affinity purification, and immunoprecipitation. The system enables high stringency wash conditions in affinity purification and immunoprecipitation experiments, facilitating efficient and specific isolation of TCF19 and its binding partners .

Additionally, the biotin-streptavidin system can be coupled with various detection methods by conjugating streptavidin to fluorescent dyes or reporter enzymes such as HRP or AP, allowing for further signal enhancement through catalyzed conversion of chromogenic, fluorescent, or chemiluminescent substrates .

How does biotin-streptavidin detection compare with other detection systems for TCF19?

When comparing biotin-streptavidin detection systems with other antibody labeling approaches for TCF19 research, several important distinctions emerge:

Signal Amplification Capacity:
The biotin-streptavidin system offers superior signal amplification compared to directly labeled antibodies. Each biotinylated antibody carries multiple biotin molecules, and each streptavidin molecule can bind four biotin molecules. This creates a significant multiplier effect that enables detection of low-abundance TCF19, especially in tissues where expression may be limited or in early developmental stages .

Sensitivity vs. Complexity Trade-off:
While direct labeling methods (antibodies directly conjugated to fluorophores or enzymes) offer simpler protocols, they typically provide lower sensitivity than biotin-streptavidin systems. For detecting TCF19 in contexts where it may be minimally expressed, such as during specific cell cycle phases or in certain tissue types, the additional sensitivity of biotin-streptavidin may be critical despite the more complex protocol .

Cost Considerations:
Biotin-conjugated detection systems generally involve more complex protocols with higher reagent costs. Researchers should consider whether TCF19 expression levels in their experimental model are sufficient for detection with simpler labeled secondary antibody approaches before implementing a biotin-streptavidin system .

Background and Specificity:
For TCF19 detection in tissues with high endogenous biotin (such as liver, kidney, and adipose tissue), specialized blocking steps are required to prevent non-specific signal when using biotin-streptavidin systems. In contrast, directly labeled antibodies avoid this potential source of background .

What are the optimal parameters for using biotin-conjugated TCF19 antibodies in immunohistochemistry?

Successful immunohistochemical detection of TCF19 using biotin-conjugated antibodies requires careful optimization of several parameters:

Antigen Retrieval:
TCF19 detection often benefits from heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). For formalin-fixed, paraffin-embedded tissues, heating at 95-100°C for 15-20 minutes typically provides optimal antigen retrieval while preserving tissue morphology .

Blocking Endogenous Biotin:
This critical step is essential for accurate TCF19 detection, particularly in biotin-rich tissues like liver, kidney, and pancreas. A sequential blocking approach is recommended:

• Block endogenous peroxidase with 0.3% H₂O₂ in methanol (10 minutes)

• Apply avidin block (15 minutes)

• Apply biotin block (15 minutes)

• Block with serum-free protein block (30 minutes)

Antibody Dilution and Incubation:
Based on available research, biotinylated TCF19 antibodies typically require optimization within the 1:200 to 1:500 dilution range. Optimal results are often achieved with overnight incubation at 4°C, which balances sensitivity with specificity .

Detection System:
For visualization, streptavidin conjugated to HRP or AP provides reliable results. Signal development using 3,3'-diaminobenzidine (DAB) typically yields optimal results for brightfield microscopy, while streptavidin conjugated to fluorophores enables multiplexed fluorescence imaging with other markers .

Counterstaining Considerations:
When examining TCF19's nuclear localization, light hematoxylin counterstaining (1-2 minutes) provides optimal contrast without obscuring the primary signal .

How should biotin-conjugated TCF19 antibodies be validated for specificity?

Thorough validation of biotin-conjugated TCF19 antibodies requires a multi-faceted approach to ensure experimental reliability:

Positive and Negative Controls:

• Positive tissue controls: Pancreatic islets and proliferating hepatocytes demonstrate reliable TCF19 expression

• Negative tissue controls: Fully differentiated adipocytes typically show minimal TCF19 expression

• Technical negative controls: Omission of primary antibody while maintaining all other steps in the protocol

• Competing peptide controls: Pre-incubation of the antibody with excess TCF19 immunizing peptide should abolish specific staining

Western Blot Validation:
Confirmation of antibody specificity by western blot should reveal a distinct band at approximately 41-45 kDa (depending on post-translational modifications). Multiple bands may indicate non-specific binding or degradation products. Lysates from tissues or cell lines with confirmed TCF19 expression (such as pancreatic β-cell lines or hepatocellular carcinoma cell lines like HepG2) serve as appropriate validation material .

siRNA Knockdown Validation:
Comparing staining patterns between TCF19 siRNA-treated cells and control siRNA-treated cells provides compelling evidence of antibody specificity. Effective TCF19 knockdown should result in substantially reduced signal intensity when using a specific antibody .

Cross-Reactivity Testing:
When examining TCF19 across multiple species, sequence alignment analysis should be performed to predict potential cross-reactivity. Experimental validation across species should be conducted even when vendors claim cross-reactivity .

What protocol modifications are needed when using biotin-conjugated TCF19 antibodies for co-immunoprecipitation studies?

Optimizing co-immunoprecipitation (co-IP) protocols for TCF19 using biotin-conjugated antibodies requires specific modifications to leverage the biotin-streptavidin interaction while minimizing potential artifacts:

Pre-clearing Optimization:
To reduce non-specific binding, pre-clear lysates using streptavidin beads before adding the biotinylated TCF19 antibody. This step is particularly important when working with tissues that express high levels of endogenous biotin-containing proteins, such as liver or pancreatic samples .

Antibody Binding Strategy:
For maximum flexibility in elution conditions, a sequential approach is recommended:

• First bind the non-biotinylated TCF19 antibody to protein A/G beads

• Then add the target lysate to capture TCF19 and its interaction partners

• This approach avoids the extremely strong biotin-streptavidin interaction that would make specific elution difficult

Elution Conditions:
If using biotinylated TCF19 antibodies with streptavidin beads directly, harsh elution conditions are required:

• Boiling in 2% SDS buffer (95°C for 5 minutes)

• Competitive elution with excess free biotin is generally ineffective due to the high affinity of the biotin-streptavidin interaction

• For native elution (to preserve protein activity), consider using cleavable biotin derivatives that allow for mild elution conditions

Control for Streptavidin-Binding Proteins:
Include appropriate controls to distinguish true TCF19 interaction partners from proteins that may bind non-specifically to streptavidin:

• Parallel IP using non-biotinylated TCF19 antibody with protein A/G beads

• Mock IP using biotinylated isotype control antibody

• Analysis of pre-clearing beads to identify common non-specific binders

How can biotin-conjugated TCF19 antibodies be utilized for chromatin immunoprecipitation (ChIP) studies?

Chromatin immunoprecipitation using biotin-conjugated TCF19 antibodies requires specialized optimization to identify TCF19 genomic binding sites:

Crosslinking Optimization:
Since TCF19 functions as a transcriptional regulator with PHD finger interaction with H3K4me3, a dual crosslinking approach often yields superior results:

• Primary formaldehyde crosslinking (1% for 10 minutes at room temperature)

• Sequential disuccinimidyl glutarate (DSG) crosslinking (2 mM for 30 minutes) before formaldehyde

• This dual approach better preserves protein-protein interactions within TCF19-containing complexes

Sonication Parameters:
Careful optimization of chromatin fragmentation is critical for successful TCF19 ChIP:

• Target fragment size: 200-500 bp

• Typical conditions: 10-12 cycles of 30 seconds ON/30 seconds OFF at 40% amplitude

• Fragment size verification by agarose gel electrophoresis is essential before proceeding

Antibody Binding Strategy:
For TCF19 ChIP, a sequential capture approach often yields the best results:

• Pre-bind biotinylated TCF19 antibody to chromatin

• Capture the antibody-chromatin complexes using streptavidin magnetic beads

• This approach minimizes background compared to direct capture with antibody-conjugated beads

PCR Primer Design for TCF19 Target Validation:
Based on existing research, primers targeting the promoter regions of TCF19-regulated genes should be designed:

• CCND1 promoter (proven TCF19 binding site)

• HDAC1 promoter (proven TCF19 binding site)

• FGF2 promoter (proven TCF19 binding site)

ChIP-Seq Considerations:
For genome-wide analysis of TCF19 binding sites, additional considerations include:

• Input normalization using 10% of pre-immunoprecipitated chromatin

• Sequencing depth: minimum 20 million uniquely mapped reads

• Peak calling algorithms optimized for transcription factors (e.g., MACS2)

What approaches can be used to study TCF19 protein-protein interactions using biotin-conjugated antibodies?

Investigating TCF19 protein interaction networks using biotin-conjugated antibodies offers several strategic advantages:

BioID Proximity Labeling:
This powerful approach involves:

• Creating a fusion protein of TCF19 with a biotin ligase (BirA*)

• Expression in cells leads to biotinylation of proteins in close proximity to TCF19

• Streptavidin pulldown followed by mass spectrometry identifies the TCF19 interactome

• This method is particularly valuable for identifying transient or weak interactions that might be missed in traditional co-IP experiments

Sequential Co-IP for Complex Composition Analysis:
For detailed analysis of TCF19-containing complexes:

• First immunoprecipitation with biotin-conjugated TCF19 antibody

• Elution under native conditions

• Second immunoprecipitation with antibodies against suspected interaction partners

• This approach confirms direct vs. indirect interactions within a complex

FRET-Based Interaction Analysis:
For live-cell imaging of TCF19 interactions:

• Express TCF19 fused to a fluorescent protein (e.g., CFP)

• Express potential interaction partner fused to a complementary fluorescent protein (e.g., YFP)

• Biotin-conjugated TCF19 antibodies can be used for validation in fixed cells

• This approach provides spatial and temporal information about TCF19 interactions

Mass Spectrometry Analysis Protocol for TCF19 Interactome:

Based on published literature, expected TCF19 interaction partners include histone-modifying enzymes and cell cycle regulators, given its role in chromatin binding via H3K4me3 recognition .

How can biotin-conjugated TCF19 antibodies be used to study its role in pancreatic β-cell function?

Investigating TCF19's critical role in pancreatic β-cell maintenance and proliferation using biotin-conjugated antibodies enables several specialized research approaches:

Multiplexed Immunofluorescence Profiling:
Biotin-conjugated TCF19 antibodies enable detailed co-expression analysis in pancreatic islets:

• Use streptavidin conjugated to a far-red fluorophore to detect biotinylated TCF19 antibody

• Combine with direct-labeled antibodies against insulin, glucagon, and cell cycle markers

• This multiplexed approach reveals TCF19 expression patterns specifically in β-cells versus α-cells

• Quantitative analysis of nuclear TCF19 levels correlates with proliferation status of β-cells

Laser Capture Microdissection with Immunohistochemical Guidance:
For isolation of TCF19-expressing β-cells:

• Perform rapid IHC staining using biotin-conjugated TCF19 antibody and streptavidin-HRP

• Use TCF19 staining pattern to guide laser capture microdissection

• Perform downstream molecular analyses (RNA-seq, proteomics) on isolated TCF19-high versus TCF19-low β-cell populations

• This approach enables molecular characterization of β-cells based on TCF19 expression levels

Flow Cytometry Sorting of TCF19-Expressing β-Cells:
For functional studies on TCF19-high versus TCF19-low β-cells:

• Disperse pancreatic islets into single cells

• Surface stain for β-cell markers

• Fix, permeabilize, and stain with biotin-conjugated TCF19 antibody

• Detect with streptavidin-fluorophore conjugate

• FACS-sort TCF19-high and TCF19-low β-cell populations

• Perform functional assays (glucose-stimulated insulin secretion, proliferation assays) on sorted populations

Examining TCF19 Expression in Diabetes Models:
Based on TCF19's association with type 1 diabetes susceptibility, comparative analysis across disease models is highly informative:

These approaches provide mechanistic insights into how TCF19 regulates β-cell mass, a critical factor in both type 1 and type 2 diabetes pathogenesis .

What are the most common technical issues when using biotin-conjugated TCF19 antibodies and how can they be resolved?

Researchers working with biotin-conjugated TCF19 antibodies frequently encounter several technical challenges that require systematic troubleshooting:

High Background in Immunohistochemistry/Immunofluorescence:

Weak or Absent TCF19 Signal:

Western Blot Detection Issues:

The biotin-streptavidin system can amplify both specific signal and background, making optimization critical for accurate TCF19 detection .

How should researchers interpret seemingly contradictory TCF19 expression data from different detection methods?

Researchers frequently encounter discrepancies in TCF19 detection across different methodologies, requiring careful interpretation:

Discrepancies Between Western Blot and Immunohistochemistry:
When western blot suggests high TCF19 expression but immunohistochemistry shows limited staining (or vice versa), consider:

• Cell-type specificity: TCF19 may be expressed in specific cell populations that are diluted in whole-tissue lysates

• Subcellular localization: TCF19 can shuttle between nucleus and cytoplasm depending on cell cycle stage

• Epitope accessibility: Different fixation methods may mask or expose different TCF19 epitopes

• Detection sensitivity threshold: Western blot and IHC have different detection limits

Quantification Considerations:
When quantifying TCF19 expression across different methods, normalize appropriately:

Physiological vs. Pathological Expression:
TCF19 expression is highly context-dependent:

• In normal tissues: TCF19 expression correlates with proliferative status and cell cycle phase

• In disease states: TCF19 may be dysregulated and expression patterns altered

• Under stress conditions: ER stress modulates TCF19 expression and localization

Cross-Validation Strategy:
To resolve contradictory data, implement a cross-validation approach:

• Use multiple antibodies targeting different TCF19 epitopes

• Compare protein-level detection with mRNA expression (RT-qPCR, RNA-seq)

• Validate with genetic approaches (siRNA knockdown, CRISPR knockout)

• Consider the temporal dimension of TCF19 expression, which fluctuates with cell cycle

What considerations are important when analyzing TCF19 expression in disease models using biotin-conjugated antibodies?

Accurate analysis of TCF19 expression in disease models requires careful attention to several critical factors:

Disease-Specific Tissue Alterations:
Pathological conditions introduce tissue changes that can affect antibody performance:

• Increased tissue autofluorescence in fibrotic or inflammatory conditions

• Altered antigen accessibility due to extracellular matrix deposition

• Increased endogenous biotin in certain pathological states

• Changes in tissue permeability that affect antibody penetration

Standardization for Cross-Model Comparison:
When comparing TCF19 expression across different disease models:

• Process all tissues simultaneously using identical protocols

• Include internal reference standards on each slide

• Use automated image acquisition with fixed exposure settings

• Implement blind scoring by multiple observers

• Normalize TCF19 expression to appropriate cell-type markers

Context-Dependent TCF19 Function:
Consider the biological context when interpreting TCF19 expression data:

Technical Optimizations for Disease Tissues:
Disease tissues often require specific protocol adjustments:

• For fibrotic tissues: Extended protease digestion (5-10 minutes with proteinase K)

• For fatty tissues: Additional deparaffinization steps and longer permeabilization

• For inflamed tissues: More stringent blocking (10% normal serum plus 1% BSA)

• For necrotic regions: Careful region-of-interest selection to avoid non-specific binding

Correlative analysis combining TCF19 expression with disease markers provides the most informative results, particularly when examining temporal changes during disease progression .

How does TCF19's role in epigenetic regulation through H3K4me3 binding inform antibody-based research approaches?

The discovery that TCF19 binds to trimethylated lysine 4 of histone H3 (H3K4me3) through its PHD finger domain has significant implications for antibody-based research strategies:

Co-Localization Studies with Epigenetic Marks:
Biotin-conjugated TCF19 antibodies can be paired with antibodies against H3K4me3 and other histone modifications to analyze the epigenetic landscape at TCF19 binding sites. Sequential chromatin immunoprecipitation (Re-ChIP) approaches using biotin-conjugated TCF19 antibodies followed by H3K4me3 antibodies can identify genomic loci where both factors co-occur. The strong biotin-streptavidin interaction facilitates stringent washing conditions necessary for Re-ChIP applications .

Functional Domain-Specific Antibodies:
The critical role of residue W316 in the PHD finger of TCF19 for H3K4me3 binding suggests the value of developing domain-specific antibodies:

• Antibodies specific to the PHD finger domain (amino acids 302-360)

• Antibodies that specifically recognize the TCF19-H3K4me3 complex

• Antibodies that distinguish between different TCF19 conformational states upon chromatin binding
  These specialized reagents would provide deeper insights into TCF19's regulatory mechanisms .

Chromatin State Analysis:
TCF19's binding to H3K4me3, a mark associated with active gene promoters, indicates its involvement in specific chromatin contexts. Antibody-based approaches can explore:

• How TCF19 binding correlates with chromatin accessibility (using techniques like ATAC-seq)

• Whether TCF19 competes with other H3K4me3 readers

• How cell cycle progression affects TCF19 chromatin occupancy

Disease-Relevant Epigenetic Alterations:
In disease contexts, epigenetic landscapes change dramatically, affecting TCF19 function:

• In type 1 diabetes, altered H3K4me3 patterns may affect TCF19 distribution

• In hepatocellular carcinoma, global increases in H3K4me3 may enhance TCF19 oncogenic function

• Biotin-conjugated TCF19 antibodies can track these disease-specific alterations

What are the emerging applications for biotin-conjugated TCF19 antibodies in single-cell analysis technologies?

Biotin-conjugated TCF19 antibodies are increasingly valuable in the rapidly evolving field of single-cell analysis:

Single-Cell Protein Analysis:
Mass cytometry (CyTOF) applications:

• Metal-tagged streptavidin detection of biotin-conjugated TCF19 antibodies

• Integration into panels with 30+ other cellular markers

• Correlation of TCF19 levels with cell cycle status at single-cell resolution

• This approach provides unprecedented resolution of TCF19 expression heterogeneity across cell populations

Spatial Transcriptomics Integration:
Combining biotin-conjugated TCF19 antibody staining with spatial transcriptomics:

• Perform multiplexed immunofluorescence with biotin-conjugated TCF19 antibody

• Overlay with spatial transcriptomics data from adjacent sections

• Correlate TCF19 protein localization with gene expression patterns

• This integration reveals spatial relationships between TCF19-expressing cells and their transcriptional environment

Microfluidic Single-Cell Western Blotting:
Emerging microfluidic platforms enable western blot analysis at single-cell level:

• Capture individual cells in microwells

• Perform in situ lysis, protein separation, and blotting

• Detect TCF19 using biotin-conjugated antibodies and fluorescent streptavidin

• This approach provides size-based confirmation of TCF19 protein along with quantitative expression data at single-cell resolution

Single-Cell Multi-Omics Applications:
Integration of biotin-conjugated TCF19 antibodies in multi-omics workflows:

These emerging technologies offer unprecedented insights into TCF19 biology at the single-cell level, revealing functional heterogeneity that would be masked in bulk analyses .

What potential exists for developing therapeutic approaches targeting TCF19 and how might biotin-conjugated antibodies facilitate this research?

The emerging understanding of TCF19's role in diverse pathological conditions suggests therapeutic potential that can be explored using biotin-conjugated antibodies:

Target Validation for Drug Development:
Biotin-conjugated TCF19 antibodies enable critical target validation steps:

• Precise tissue and subcellular localization in disease models

• Confirmation of target engagement using competitive binding assays

• Monitoring of protein expression changes in response to candidate therapeutics

• These applications support go/no-go decisions in early drug development pipelines

Antibody-Drug Conjugate (ADC) Development:
For conditions with TCF19 overexpression (such as hepatocellular carcinoma):

• Biotin-conjugated TCF19 antibodies can be used to evaluate internalization kinetics

• The biotin-streptavidin system allows modular attachment of various cytotoxic payloads

• This modular approach enables rapid screening of multiple therapeutic configurations

• Biotinylated antibodies facilitate proof-of-concept studies before investing in direct conjugation

Small Molecule Inhibitor Discovery:
For targeting the TCF19-H3K4me3 interaction:

• Development of competition assays using biotin-conjugated TCF19 antibodies

• High-throughput screening for compounds that disrupt TCF19 chromatin binding

• Structure-activity relationship studies guided by changes in TCF19 localization

• These applications accelerate the identification of lead compounds that modulate TCF19 function

Therapeutic Delivery Strategies:
Leveraging the biotin-avidin system for targeted delivery:

• Biotinylated TCF19-targeting antibodies can be paired with avidin-conjugated nanoparticles

• This modular approach allows delivery of various therapeutic payloads

• The approach can be optimized for specific tissue targeting

• These strategies are particularly valuable for delivering RNA therapeutics targeting TCF19 expression

Biomarker Development:
TCF19 expression patterns as predictive or prognostic biomarkers:

• In type 1 diabetes: TCF19 expression in residual β-cells may predict disease progression

• In hepatocellular carcinoma: TCF19 levels may predict response to specific therapies

• Biotin-conjugated antibodies enable sensitive detection in limited clinical samples

• Standardized immunoassays using biotin-streptavidin detection support clinical validation studies