TCF19 Antibody, HRP conjugated

What is TCF19 and what cellular roles make it significant for antibody-based detection?

TCF19 (Transcription Factor 19) is a 345 amino acid nuclear protein characterized by one forkhead-associated (FHA) domain, a proline-rich domain, and one PHD-type zinc finger. It functions as a growth-regulated trans-activating factor involved in transcription of genes governing late stages of cell cycle progression, particularly G1 to S phase transition and entry into G2 and mitosis . This protein's expression notably increases during the G1-S phase of the cell cycle, highlighting its importance in regulating cellular proliferation .

The protein's role in cell cycle regulation makes it particularly valuable for studying proliferation mechanisms in both normal and pathological conditions. Researchers targeting TCF19 should note its nuclear localization when designing experiments, as this impacts fixation protocols and permeabilization requirements for successful antibody binding.

What are the optimal applications for HRP-conjugated TCF19 antibodies?

HRP-conjugated TCF19 antibodies are particularly effective for several applications:

• ELISA: HRP-conjugated antibodies can be used at dilutions of 1:5000-1:10000 for optimal signal-to-noise ratio .

• Western Blotting: The HRP conjugation eliminates the need for secondary antibody incubation, reducing background and cross-reactivity issues.

• Immunohistochemistry: For IHC applications, optimal dilutions are typically 1:50-1:100, allowing for direct visualization of TCF19 expression patterns in tissue sections .

When selecting application-specific protocols, researchers should note that TCF19 antibodies have been validated for multiple applications including western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) .

What species cross-reactivity should researchers consider when selecting TCF19 antibodies?

TCF19 antibodies exhibit varying species cross-reactivity profiles depending on the specific clone and production method. Based on current available antibodies:

When working with non-human models, it's crucial to select antibodies with demonstrated cross-reactivity to your species of interest. For comparative studies across multiple species, select antibodies with broad reactivity or use species-specific antibodies with similar epitope recognition .

How does HRP conjugation affect storage conditions and shelf-life of TCF19 antibodies?

HRP-conjugated antibodies require specific storage protocols to maintain enzymatic activity:

• Temperature: Store at 2-8°C for short-term use (1-2 weeks) and at -20°C for long-term storage.

• Buffer conditions: HRP conjugates should be stored in buffers containing stabilizers and preservatives.

• Aliquoting: Divide into single-use aliquots to prevent repeated freeze-thaw cycles that can damage both the antibody and the HRP enzyme.

• Light sensitivity: Protect from light to prevent photobleaching of the chromogenic components.

When working with HRP-conjugated TCF19 antibodies, include positive controls in each experiment to verify enzymatic activity, especially after extended storage periods.

How can TCF19 antibodies be utilized in cancer immunotherapy research?

TCF19 has emerging significance in cancer immunotherapy research, requiring specific methodological approaches:

• Expression analysis: Use TCF19 antibodies in immunohistochemistry to correlate expression with immunotherapy response. Studies have identified differences in TCF19 expression between immunotherapy responders and non-responders .

• Methodology for response evaluation:

  • Categorize patients into responder groups (complete response/partial response) versus non-responders (stable disease/progressive disease)

  • Use Wilcoxon rank-sum test to investigate expression differences between groups

  • Correlate TCF19 expression with clinical outcomes

• Drug sensitivity correlations: Analyze TCF19 expression in relation to drug sensitivity using datasets like NCI-60. This requires:

  • RNA-seq expression data collection

  • Drug sensitivity profiles

  • Correlation analysis with statistical thresholds (p<0.05)

For such studies, carefully validated antibodies with confirmed specificity are essential to ensure accurate representation of TCF19 expression levels in clinical samples.

What are optimal western blot protocols for detecting TCF19 using HRP-conjugated antibodies?

Detecting TCF19 (approximately 37-42 kDa) using HRP-conjugated antibodies requires specific optimization:

• Sample preparation:

  • For nuclear proteins like TCF19, use nuclear extraction buffers containing protease inhibitors

  • Sonicate briefly to shear genomic DNA that can interfere with protein migration

  • Heat samples at 95°C for 5 minutes in reducing buffer

• Gel parameters:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Load 20-50 μg of total protein per lane

• Transfer conditions:

  • Wet transfer at 100V for 60-90 minutes or 30V overnight at 4°C

  • Use PVDF membranes for better protein retention

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour

  • Dilute HRP-conjugated TCF19 antibody to 1:1000-1:5000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) substrates

  • For low abundance detection, consider using ECL substrates with extended signal duration

When troubleshooting, include positive control lysates from cells known to express TCF19, particularly those in G1-S transition phase when TCF19 expression peaks .

How should researchers optimize immunohistochemistry protocols for TCF19 detection in tissue samples?

Optimizing IHC protocols for TCF19 detection requires attention to several methodological aspects:

• Antigen retrieval methods:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Pressure cooker treatment for 20 minutes followed by 20 minutes cooling

• Blocking endogenous peroxidase and biotin:

  • 3% hydrogen peroxide for 10 minutes

  • Avidin/biotin blocking kit if using biotin-based detection systems

• Antibody dilution optimization:

  • For HRP-conjugated TCF19 antibodies, start with 1:50-1:100 dilution

  • Optimize through titration experiments for each tissue type

• Visualization considerations:

  • DAB substrate incubation for 2-10 minutes (monitor microscopically)

  • Counterstain with hematoxylin for 30-60 seconds

• Controls to include:

  • Positive control tissues (skin samples for psoriasis studies, proliferating tissues)

  • Negative controls (antibody diluent only)

  • Isotype controls to rule out non-specific binding

For FFPE tissues, perform additional validation using alternative detection methods (like IF or WB) to confirm specificity, as fixation can modify epitopes and affect antibody recognition.

What methodological approaches resolve contradictory TCF19 expression data across different experimental platforms?

When faced with contradictory TCF19 expression data, implement these methodological approaches:

• Cross-platform validation:

  • Validate protein expression (antibody-based) with mRNA expression data

  • Use multiple antibody clones targeting different epitopes

  • Compare monoclonal vs. polyclonal antibody results

• Biological context analysis:

  • Assess cell cycle phase - TCF19 expression peaks during G1-S transition

  • Consider tissue-specific expression patterns

  • Evaluate potential post-translational modifications affecting antibody binding

• Technical validation:

  • Perform spike-in experiments with recombinant TCF19

  • Use siRNA/shRNA knockdown of TCF19 to confirm specificity

  • Conduct immunoprecipitation followed by mass spectrometry

• Data integration methodology:

  • Use Gene Set Variation Analysis (GSVA) to move from gene-level to pathway-level analysis

  • Apply statistical methods accounting for batch effects and platform differences

  • Implement multivariate analysis to identify confounding variables

How can researchers utilize TCF19 antibodies in studying its potential role in psoriasis pathogenesis?

The TCF19 gene is situated in a critical region on chromosome 6 linked to psoriasis vulgaris, suggesting its involvement in this hyperproliferative skin disorder . Methodological approaches include:

• Comparative expression analysis:

  • Use HRP-conjugated TCF19 antibodies (1:50-1:100 dilution) for IHC on psoriatic vs. normal skin sections

  • Quantify nuclear staining intensity using digital image analysis

  • Correlate with clinical disease severity measures

• Functional studies methodology:

  • Co-stain with proliferation markers (Ki-67, PCNA) to establish correlation with hyperproliferation

  • Perform cell cycle analysis in keratinocytes with manipulation of TCF19 expression

  • Use chromatin immunoprecipitation (ChIP) to identify TCF19 target genes in keratinocytes

• Genetic association validation:

  • Correlate TCF19 protein expression with known genetic variants

  • Implement case-control studies with immunohistochemical analysis

  • Perform genotype-phenotype correlation analyses

These methodological approaches can help elucidate how TCF19's regulatory effects on late cell cycle-specific genes contribute to the hyperproliferative phenotype characteristic of psoriasis.

What are common technical issues when using HRP-conjugated TCF19 antibodies and their solutions?

When working with HRP-conjugated TCF19 antibodies, remember they contain sodium azide, which is a hazardous substance requiring proper handling by trained personnel .

How can researchers validate TCF19 antibody specificity for immunotherapy studies?

For immunotherapy studies, rigorous validation of TCF19 antibodies is crucial:

• Pre-absorption controls:

  • Incubate antibody with excess recombinant TCF19 protein (amino acids 12-250)

  • Parallel testing of pre-absorbed and non-pre-absorbed antibody should show elimination of specific signal

• Genetic validation:

  • Test on TCF19 knockout/knockdown samples

  • Use cell lines with verified TCF19 expression levels as positive and negative controls

• Correlation with genomic data:

  • Verify antibody results against TCF19 mRNA expression data from TCGA database

  • Implement RNA-seq validation in parallel with protein detection methods

• Multi-antibody approach:

  • Compare results using antibodies targeting different epitopes

  • Test both polyclonal and monoclonal antibodies with different host origins

• Functional validation:

  • Correlate staining pattern with known biological functions of TCF19

  • Verify nuclear localization consistent with its role as a transcription factor

In immunotherapy studies where accurate quantification is critical, implement at least three different validation approaches to ensure reliable results.

What controls should be included when studying TCF19 in drug sensitivity assays?

When studying TCF19 in relation to drug sensitivity, implement these essential controls:

• Positive controls:

  • Cell lines with verified high TCF19 expression (preferably synchronized in G1-S phase)

  • Recombinant TCF19 protein standards for quantitative assays

• Negative controls:

  • TCF19 knockdown/knockout cell lines

  • Cell cycle-arrested cells (G0/G1) where TCF19 expression should be minimal

  • Isotype control antibodies to assess non-specific binding

• Experimental controls:

  • Vehicle-only treatments for drug studies

  • Time-course controls to account for cell cycle fluctuations in TCF19 expression

  • Multi-dose response curves to establish proper dynamic range

• Analytical controls:

  • Include reference genes/proteins with stable expression

  • Use parallel alternative methods for TCF19 detection (e.g., WB and qPCR)

  • Apply statistical methods appropriate for pharmacological studies (IC50 calculations)

For NCI-60 based drug sensitivity correlations, implement correlation analysis with a statistical threshold of p<0.05 to identify significant associations between TCF19 expression and drug response .

How can TCF19 antibodies be applied in multi-parameter flow cytometry protocols?

Implementing TCF19 in multi-parameter flow cytometry requires specific methodological considerations:

• Sample preparation protocol:

  • Fix cells with 4% paraformaldehyde for 15 minutes

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

  • For multi-parameter analysis, use sequential staining approach

• Panel design considerations:

  • Pair TCF19 with cell cycle markers (Ki-67, PCNA, cyclins)

  • Include cell surface markers relevant to your cell type of interest

  • Design compensation matrix accounting for HRP emission spectrum

• Signal amplification strategies:

  • For low expression detection, implement tyramide signal amplification

  • Use fluorescent substrates compatible with HRP for direct detection

  • Consider alternative conjugates (PE, FITC, Alexa Fluor) available for TCF19 antibodies

• Gating strategy optimization:

  • Gate on cell cycle phases to correlate with TCF19 expression

  • Implement FMO (fluorescence minus one) controls for accurate gating

  • Use bivariate plots of TCF19 versus cell cycle markers

This approach allows for quantitative single-cell analysis of TCF19 expression in relation to cell cycle status and other cellular parameters.

What methodologies enable integration of TCF19 expression data with immunotherapy response prediction?

To effectively integrate TCF19 expression data with immunotherapy response prediction:

These methodological approaches can help establish whether TCF19 expression serves as a legitimate biomarker for immunotherapy response prediction in clinical settings.

What technological advances might improve TCF19 detection sensitivity and specificity?

Emerging technologies poised to enhance TCF19 detection include:

• Next-generation antibody engineering:

  • Single-domain antibodies with improved tissue penetration

  • Recombinant antibody fragments with enhanced epitope specificity

  • Site-specific conjugation technologies for better HRP attachment

• Advanced detection systems:

  • Proximity ligation assays for detecting TCF19 protein interactions

  • Digital ELISA platforms with single-molecule detection capability

  • Multiplexed ion beam imaging (MIBI) for subcellular localization studies

• Computational approaches:

  • Machine learning algorithms for automated signal quantification

  • Integrative analysis frameworks combining protein, mRNA, and genetic data

  • 3D reconstruction of TCF19 subcellular distribution

• Novel substrate technologies:

  • Photoswitchable HRP substrates for controlled visualization

  • Quantum dot-coupled detection systems for enhanced sensitivity

  • Lanthanide-based time-resolved fluorescence for background reduction

These technological advances will enable more precise quantification of TCF19 expression patterns in both research and clinical applications.

How might TCF19 antibodies contribute to understanding its role in cellular proliferation beyond cancer?

TCF19's involvement in cell cycle regulation suggests broader applications beyond cancer research:

• Stem cell biology applications:

  • Track TCF19 expression during differentiation processes

  • Correlate with stemness markers in pluripotent and tissue-specific stem cells

  • Investigate role in self-renewal versus differentiation decisions

• Developmental biology methods:

  • Implement lineage tracing experiments with TCF19 as a proliferation marker

  • Study temporal expression patterns during organogenesis

  • Correlate with developmental timing of proliferative events

• Regenerative medicine approaches:

  • Monitor TCF19 in tissue regeneration models

  • Correlate expression with healing rates in wound models

  • Investigate potential as a biomarker for regenerative capacity

• Aging research methodologies:

  • Compare TCF19 expression patterns across age groups

  • Correlate with senescence markers in aging tissues

  • Investigate epigenetic regulation of TCF19 during aging

These applications would benefit from carefully validated TCF19 antibodies with demonstrated specificity across diverse experimental systems and tissue types.