TCEAL3 Antibody

What is TCEAL3 and what is its primary function in cellular processes?

TCEAL3 (Transcription Elongation Factor A (SII)-Like 3) is a member of the TFS-II family that plays a crucial role in transcription by facilitating RNA polymerase II release from transcriptional arrest. The 348-amino acid protein features one TFIIS N-terminal domain, one TFIIS central domain, and one TFIIS-type zinc finger . TCEAL3 functions by activating RNA polymerase's intrinsic RNA cleavage activity, enabling nascent transcript cleavage and new 3'-terminus formation, thus allowing transcription to resume when RNA polymerases become trapped at DNA arresting sites . Recent research has also revealed TCEAL3's unexpected role in promoting apoptosis in various cancer cell lines, suggesting its potential tumor suppressor functions .

What types of TCEAL3 antibodies are currently available for research applications?

Current research-grade TCEAL3 antibodies include:

Both unconjugated and conjugated variants are available, including HRP, FITC, PE, and Alexa Fluor® conjugates for specialized applications . Many commercial antibodies undergo rigorous validation through multiple techniques including protein arrays, western blotting, and immunohistochemistry across various tissues .

What are the optimal conditions for using TCEAL3 antibodies in immunohistochemistry (IHC)?

For optimal TCEAL3 detection in IHC applications, follow this methodology:

• Tissue preparation: Use formalin-fixed paraffin-embedded (FFPE) tissue sections (4-6 μm thickness), with antigen retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 20 minutes at 95-98°C.

• Blocking and antibody incubation: Block with 3% BSA and 5% horse serum in TBS for 60 minutes at room temperature . Incubate with primary TCEAL3 antibody at 1:50-1:200 dilution (concentration-dependent on specific antibody) overnight at 4°C .

• Detection system: For polyclonal rabbit antibodies, use either HRP-polymer or biotin-streptavidin systems with DAB as the chromogen. For mouse monoclonal antibodies, appropriate anti-mouse secondary antibodies should be used.

• Controls: Include skeletal muscle tissue as a positive control (highest endogenous TCEAL3 expression) . Recommended negative controls include either antibody diluent alone or isotype-matched control antibodies.

The subcellular localization pattern should be primarily nuclear with some cytoplasmic staining in certain cell types, particularly in epithelial cells .

How should western blot protocols be optimized for TCEAL3 detection?

Optimized western blot protocol for TCEAL3 detection:

• Sample preparation: Extract proteins using RIPA buffer supplemented with protease inhibitors. Load 20-50 μg of total protein per lane.

• Gel electrophoresis and transfer: Use 10-12% SDS-PAGE gels and wet transfer to PVDF membranes (recommended over nitrocellulose for higher sensitivity).

• Antibody incubation: Block membranes with 5% non-fat milk in TBST for 1 hour. Incubate with primary TCEAL3 antibody (1:500-1:1000 dilution) overnight at 4°C. For monoclonal antibodies like TCEA3 (C-7), 1:1000 dilution is recommended .

• Detection: Use HRP-conjugated secondary antibodies and enhanced chemiluminescence detection systems. The expected molecular weight for TCEAL3 is approximately 23 kDa .

• Validation controls: Include recombinant TCEAL3 protein as a positive control. For tissue/cell samples, skeletal muscle lysates serve as appropriate positive controls .

For challenging samples with low TCEAL3 expression, immunoprecipitation followed by western blotting can improve detection sensitivity.

What methodology should be used for studying TCEAL3 subcellular localization?

For accurate TCEAL3 subcellular localization studies:

• Immunofluorescence approach:

  • Fix cells with 4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilize with 0.1% Triton X-100 (5 minutes)

  • Block with 1% BSA in PBS (30 minutes)

  • Incubate with primary TCEAL3 antibody (1:100-1:200) overnight at 4°C

  • Visualize using fluorophore-conjugated secondary antibodies

  • Include DAPI nuclear counterstain

• Co-localization studies: Combine TCEAL3 antibody with markers for specific cellular compartments, particularly nuclear speckles (SC35) and transcription factories (RNA Polymerase II), given TCEAL3's role in transcription elongation .

• Live-cell imaging: For dynamic studies, consider using TCEAL3 antibody fragments conjugated to cell-permeable peptides or expression of fluorescently-tagged TCEAL3.

Research has shown that TCEAL3 localization can shift from cytoplasmic to nuclear during cellular differentiation , making it important to analyze multiple time points during differentiation processes. In RMS cell lines with exogenous TCEAL3 expression, the protein localizes primarily to the nucleus with some cytoplasmic distribution, particularly in ERMS cell lines .

How can TCEAL3 antibodies be utilized in chromatin immunoprecipitation (ChIP) studies?

For effective TCEAL3 ChIP experiments:

• Crosslinking optimization: Use 1% formaldehyde for 10 minutes at room temperature for protein-DNA crosslinking, followed by quenching with 125 mM glycine.

• Sonication parameters: Optimize sonication conditions to generate DNA fragments of 200-500 bp (typically 10-15 cycles of 30 seconds on/30 seconds off at medium power).

• Immunoprecipitation: Pre-clear chromatin with protein A/G beads for 1 hour. For immunoprecipitation, use 2-5 μg of TCEAL3 antibody per reaction, ideally a ChIP-validated antibody or one known to work in IP applications like the TCEA3 Antibody (C-7) AC variant .

• Controls: Include:

  • Input sample (non-immunoprecipitated chromatin)

  • IgG negative control (matching the host species of TCEAL3 antibody)

  • Positive control antibody (RNA Polymerase II)

• Data analysis: Following ChIP-seq, analyze TCEAL3 binding patterns in relation to transcription elongation sites, paused polymerase regions, and other transcription factors involved in elongation.

Given TCEAL3's role in transcription elongation, expected enrichment should occur at gene bodies rather than promoter regions, with potential concentration at sites known to cause transcriptional pausing or arrest.

What approaches can be used to study TCEAL3's role in apoptosis in different cancer contexts?

To investigate TCEAL3's apoptotic functions in cancer research:

• Expression analysis methodology:

  • Analyze TCEAL3 expression across cancer datasets using immunohistochemistry with appropriate antibodies

  • Compare expression with patient survival data as shown in previous studies where higher TCEAL3 expression correlated with better five-year survival rates

• Functional studies:

  • Ectopic expression system: Transfect cancer cell lines with TCEAL3 expression vectors followed by:

    • TUNEL assays to detect DNA fragmentation

    • Annexin V/PI flow cytometry to quantify early/late apoptotic cells

    • Western blotting for apoptotic markers (cleaved caspases 3, 8, 9)

  • Pathway analysis: Use specific caspase inhibitors (intrinsic, extrinsic, or pan-caspase) to determine which apoptotic pathway is activated by TCEAL3

• Mechanistic investigations:

  • Co-immunoprecipitation with TCEAL3 antibodies to identify protein interaction partners

  • RNA-seq after TCEAL3 modulation to identify transcriptional targets

Previous research has demonstrated that TCEAL3 promotes apoptosis through both intrinsic and extrinsic pathways, with cleaved products of caspase-3, caspase-8, and caspase-9 all detected in TCEAL3-overexpressing cells . Additionally, TCEAL3 expression enhances sensitivity to chemotherapeutic drugs, including TRAIL, suggesting its potential in combination therapies .

How can single-cell techniques incorporate TCEAL3 antibodies for advanced cellular profiling?

For integrating TCEAL3 into single-cell profiling workflows:

• Single-cell protein analysis:

  • Mass cytometry (CyTOF): Conjugate TCEAL3 antibodies with rare earth metals for inclusion in CyTOF panels alongside lineage markers

  • Imaging mass cytometry: Combine TCEAL3 detection with spatial context in tissue sections

• Multi-omics approach:

  • CITE-seq/REAP-seq: Use oligonucleotide-tagged TCEAL3 antibodies in combination with single-cell RNA-seq

  • Validate TCEAL3 protein levels against transcript expression, considering previous observations of RNA-protein correlation (specificity improvement seen when incorporating orthogonal proteomic data)

• Panel design considerations:

  • Apply interactive human-in-the-loop platforms like Cytomarker for optimal antibody panel design incorporating TCEAL3

  • Consider TCEAL3's expression pattern when selecting appropriate tissue controls

Recent validation approaches have demonstrated that scRNA-seq data can predict cell-type specific highly expressed markers in CyTOF single-cell antibody screens with high positive predictive value and sensitivity . For TCEAL3, tissue-specific expression patterns and cancer-related downregulation should be considered when designing experimental strategies and interpreting results.

What are common issues with TCEAL3 antibodies and how can they be addressed?

Common challenges with TCEAL3 antibody applications and their solutions:

For particularly challenging applications, consider using monoclonal antibodies like TCEA3 (C-7) that offer high specificity, or antibodies with extensive validation documented across multiple applications .

How should researchers approach contradictory results when studying TCEAL3 expression or function?

When facing contradictory results in TCEAL3 research:

• Antibody validation assessment:

  • Compare epitope regions targeted by different antibodies

  • Verify specificity through protein arrays (like the 364-protein arrays used for Prestige Antibodies)

  • Consider antibodies with multiple validation methods (IHC, WB, IF)

• Context-dependent expression analysis:

  • TCEAL3 shows tissue-specific expression patterns

  • Expression can be regulated by methylation (correlation with DNA methylation in cancer cell lines)

  • Consider developmental stage and differentiation status (TCEAL3 translocation from cytoplasm to nucleus during differentiation)

• Methodological reconciliation:

  • Compare antibody-based detection with transcript-level data

  • Acknowledge the correlation limitations between RNA and protein levels (correlations between 0.38 and 0.58 reported in immune cell types)

  • Apply orthogonal validation approaches combining multiple techniques

• Biological interpretations:

  • Consider the dual functions of TCEAL3 in transcription elongation and apoptosis regulation

  • Recognize potential cell-type specific functions and effects

When publishing contradictory findings, clearly document the specific antibodies used, their validation status, and the experimental conditions to facilitate proper interpretation and reproducibility.

What considerations are important when interpreting TCEAL3 localization data in different cell types?

Key considerations for interpreting TCEAL3 localization include:

• Cell type and physiological state:

  • TCEAL3 has been shown to translocate from the cytoplasm to the nucleus during cellular differentiation

  • Different distribution patterns between ERMS and ARMS cell lines have been observed (more cytoplasmic localization in ERMS)

• Technical considerations:

  • Fixation methods can affect nuclear protein detection (cross-linking fixatives like paraformaldehyde better preserve nuclear structure)

  • Permeabilization conditions influence antibody accessibility

  • Different antibodies may recognize distinct conformational epitopes affecting localization patterns

• Co-localization analysis:

  • Correlation with RNA Polymerase II distribution patterns

  • Relationship to transcriptionally active regions

  • Association with other transcription elongation factors

• Functional context:

  • Nuclear localization correlates with TCEAL3's role in transcription elongation

  • Cytoplasmic localization may relate to alternative functions or inactive states

  • Dynamic distribution may reflect regulatory mechanisms

• Validation approaches:

  • Use multiple antibodies targeting different epitopes of TCEAL3

  • Compare with fluorescently-tagged TCEAL3 expressed at physiological levels

  • Employ super-resolution microscopy for precise subcellular localization

The predominant nuclear localization with some cytoplasmic distribution observed in experimental systems aligns with TCEAL3's primary role in nuclear transcription processes while suggesting potential additional cytoplasmic functions .

How can TCEAL3 antibodies be utilized in therapeutic antibody development?

Emerging approaches for translating TCEAL3 research into therapeutic applications:

• Antibody-based delivery systems:

  • Develop bispecific antibodies targeting TCEAL3 and cell surface markers for intracellular delivery

  • Utilize cell-penetrating antibody fragments (scFvs) targeting TCEAL3 to modulate its activity

  • Apply lessons from the NT-108 antibody development process, particularly the single-chain Fv construction that improved cryo-EM maps by preventing preferred orientations

• Cancer therapeutic potential:

  • Target pathways downstream of TCEAL3 loss in cancers

  • Design TCEAL3-based approaches to enhance sensitivity to chemotherapeutic drugs, leveraging findings that restoration of TCEAL3 expression enhanced sensitivity to chemotherapeutic drugs including TRAIL

  • Develop combination therapies targeting TCEAL3-related apoptotic pathways

• Structural biology applications:

  • Use high-affinity TCEAL3 antibodies in structural studies of transcription elongation complexes

  • Apply biophysics-informed modeling approaches (similar to those used for designing antibodies with customized specificity profiles)

• Methodological innovations:

  • Apply screening approaches similar to phage display experiments with systematic variations in CDR3 regions

  • Consider the VL-VH orientation for optimal scFv construction as demonstrated in other antibody development work

The finding that TCEAL3 expression correlates with better patient outcomes suggests potential prognostic and therapeutic value in restoring or mimicking TCEAL3 function in cancers where it is downregulated.

What novel research applications are emerging for TCEAL3 antibodies in gene regulation studies?

Cutting-edge applications of TCEAL3 antibodies in gene regulation research:

• Advanced genomic techniques:

  • CUT&RUN or CUT&Tag: Employ TCEAL3 antibodies for precise genomic localization with lower background than traditional ChIP

  • HiChIP/PLAC-seq: Investigate the role of TCEAL3 in three-dimensional chromatin organization during transcription elongation

  • CRISPR-based approaches: Combine with TCEAL3 antibodies for targeted studies of specific genomic loci

• Multi-omics integration:

  • Correlate TCEAL3 binding sites with transcriptional output

  • Analyze TCEAL3 distribution in relation to chromatin accessibility

  • Investigate relationships between TCEAL3 binding and RNA polymerase II pausing/release

• Single-molecule techniques:

  • Super-resolution microscopy to visualize TCEAL3 in transcription factories

  • Single-molecule tracking to analyze TCEAL3 dynamics during transcription elongation

  • In vitro reconstitution systems to study TCEAL3's biochemical activities

• Transcriptional arrest models:

  • Use TCEAL3 antibodies to analyze its recruitment to sites of transcriptional pausing

  • Study the interplay between TCEAL3 and other elongation factors at arrest sites

  • Investigate TCEAL3's role in handling transcription-replication conflicts

These emerging applications build upon TCEAL3's established role in transcription by facilitating RNA polymerase II release from transcriptional arrest and enabling transcription to resume through RNA cleavage activity .

How should researchers approach TCEAL3 antibody selection for innovative multi-parameter imaging studies?

For cutting-edge multi-parameter imaging with TCEAL3 antibodies:

• Antibody characteristics optimization:

  • Prioritize high-affinity antibodies with documented specificity across multiple validation methods

  • Consider directly conjugated primary antibodies to minimize background in multiplexed approaches

  • Select antibodies targeting distinct epitopes for combinatorial approaches

  • For highly multiplexed imaging, consider metal-conjugated antibodies compatible with Imaging Mass Cytometry or MIBI-TOF

• Experimental design considerations:

  • Validate antibody performance in multiplexed conditions before experimental application

  • Include appropriate controls targeting proteins with known localization patterns

  • Design panels that incorporate functional partners of TCEAL3

• Advanced imaging methodologies:

  • Cyclic immunofluorescence (t-CyCIF/4i): Use TCEAL3 antibodies in sequential staining rounds

  • Clearing-based approaches (CLARITY, CUBIC): Test TCEAL3 antibody penetration in whole-tissue imaging

  • Expansion microscopy: Validate epitope retention after sample expansion

• Data analysis approaches:

  • Apply machine learning for complex pattern recognition in TCEAL3 distribution

  • Develop quantitative metrics for co-localization with transcription machinery

  • Integrate spatial and functional data through computational modeling

A recent novel approach demonstrated the successful validation of cell surface markers predicted from scRNA-seq using CyTOF with over 3.5 million cells . Similar methods could be applied to validate TCEAL3 expression patterns in relation to cellular states and differentiation processes.