TCEA1 Antibody

What is TCEA1 and what is its primary biological function?

TCEA1, also known as TFIIS or GTF2S, functions as a transcription elongation factor essential for efficient RNA polymerase II transcription. It specifically helps overcome "arresting sites" in DNA that can trap elongating RNA polymerases in locked ternary complexes. TCEA1 stimulates the arrested polymerase to cleave the nascent transcript, allowing transcription to resume from the new 3'-terminus . Beyond this mechanical role, TCEA1 significantly influences myeloid cell development by regulating proliferation, differentiation, and survival pathways. Research has shown that TCEA1 knockdown enhances proliferation of myeloid cells while impairing their differentiation and inhibiting apoptosis .

What experimental applications have been validated for TCEA1 antibodies?

TCEA1 antibodies have been validated across multiple experimental applications with specific optimal conditions:

For optimal IHC results, antigen retrieval with TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative . When performing Western blot, many researchers use GAPDH as a loading control and 5% NFDM/TBST as blocking buffer .

How does TCEA1 affect myeloid cell development and differentiation?

TCEA1 plays a crucial regulatory role in myeloid cell development through multiple mechanisms:

TCEA1 knockdown profoundly affects myeloid differentiation by:

• Elevating expression of early neutrophil genes (myeloperoxidase, proteinase3, neutrophil elastase) that mark myeloblasts and promyelocytes

• Decreasing expression of differentiation markers including lactoferrin, gelatinase, and cysteine-rich secretory protein 3

• Altering morphology - cells with silenced TCEA1 exhibit immature features with single ovoid nuclei and high nuclear:cytoplasmic ratios

• Inhibiting apoptosis - TCEA1-silenced myeloid cells show significantly reduced apoptosis rates (16.5-22.6%) compared to control cells (54%) when cultured with G-CSF

These effects collectively contribute to the accumulation of immature myeloid cells and blockage of terminal differentiation, suggesting potential implications for leukemic transformation.

What is the relationship between TCEA1 and transcription-associated genomic instability?

TCEA1 deficiency leads to significant transcription-associated genomic instability through several interconnected mechanisms:

In Tcea1−/− cells, researchers have observed:

• Increased R-loop formation at telomeres, causally contributing to the release of telomeric DNA fragments into the cytoplasm

• Higher association of elongating RNA polymerase II (pS2-PolII) with 8-oxoG DNA lesions compared to wild-type controls

• Elevated DNA damage markers - a higher percentage of Tcea1−/− MEFs exhibit multiple foci positive for both γH2AX and 53BP1 compared to wild-type controls

• Restoration of genomic stability when R-loops are resolved - transfection of recombinant RNase H substantially lowers DNA damage markers in TCEA1-deficient cells

These findings establish TCEA1 as a critical factor in preventing R-loop-mediated genomic instability, particularly at telomeric regions, highlighting its importance in maintaining genome integrity.

How can researchers optimize TCEA1 antibody protocols for studying R-loop formation?

For investigating R-loops using TCEA1 antibodies, researchers should implement this optimized protocol sequence:

• R-loop preservation during sample preparation:

  • Fix cells with 1% formaldehyde (10 minutes at room temperature)

  • Lyse cells in buffer containing protease inhibitors

  • Carefully extract nucleic acids avoiding procedures that disrupt RNA-DNA hybrids

• DNA-RNA hybrid immunoprecipitation (DRIP):

  • Fragment chromatin via sonication (200-500bp fragments)

  • Perform immunoprecipitation with S9.6 antibody for RNA-DNA hybrids

  • Include parallel RNase H-treated samples as negative controls

• Sequential ChIP approach:

  • First ChIP: Use TCEA1 antibody (1:50 dilution for IP)

  • Second ChIP: Use S9.6 antibody on TCEA1-precipitated material

  • Compare results with single ChIP controls

• Visualization and quantification:

  • Immunofluorescence with TCEA1 antibody (1:500) and S9.6 antibody

  • Dot blot analysis of genomic DNA with S9.6 antibody

  • Quantitative PCR of regions of interest (especially telomeres)

Including RNase H treatment controls is essential for confirming R-loop specificity, as demonstrated in studies where RNase H transfection substantially reduced DNA damage markers in Tcea1−/− MEFs.

What markers should researchers monitor when studying TCEA1's impact on myeloid differentiation?

To comprehensively assess TCEA1's role in myeloid differentiation, researchers should monitor this panel of markers:

When establishing the experimental system, researchers should use validated shRNA constructs targeting TCEA1, confirm knockdown efficiency by Western blot (1:500-1:1000 dilution), and include appropriate differentiation inducers (e.g., G-CSF for granulocytic differentiation) .

How should researchers design experimental controls when using TCEA1 antibodies to study transcriptional stress?

Robust experimental controls are essential for reliable TCEA1 antibody-based studies of transcriptional stress:

• Antibody specificity controls:

  • Positive control: Validated TCEA1-expressing cells (HeLa, 293T)

  • Negative control: TCEA1 knockout/knockdown cells

  • Cross-reactivity check: Note that some antibodies (like EPR14821) recognize TCEA1, TCEA2, and TCEA3

  • Loading control validation: Document pulldown efficiency (should approach 80% for ChIP applications)

• Transcriptional stress induction controls:

  • Positive controls: Etoposide-treated cells (for DNA damage)

  • Untreated baseline controls

  • Stress recovery time course

• R-loop-specific controls:

  • RNase H-treated samples (to specifically degrade RNA in RNA:DNA hybrids)

  • Overexpression of factors that resolve R-loops (SETX, RNase H)

  • S9.6 antibody specificity validation

• TCEA1 function validation:

  • TCEA1 rescue experiments in knockout backgrounds

  • Phenotype comparison between shRNA and CRISPR-based TCEA1 depletion

  • Dose-response relationship between TCEA1 levels and observed phenotypes

These controls ensure that observed effects are specifically attributable to TCEA1 function rather than experimental artifacts or off-target effects.

What methods can researchers employ to investigate TCEA1's role in leukemia and hematopoietic malignancies?

To investigate TCEA1's potential role in leukemia and hematopoietic malignancies, researchers should apply these methodological approaches:

• Expression analysis in patient samples:

  • Compare TCEA1 expression levels between normal hematopoietic cells and leukemic cells using Western blot (1:500-1:3000 dilution)

  • Perform immunohistochemistry on bone marrow biopsies (1:20-1:200 dilution with TE buffer pH 9.0 for antigen retrieval)

  • Analyze public databases like Gene Expression Omnibus (GEO) for TCEA1 expression patterns across hematological malignancies

• Functional studies in leukemia models:

  • Establish TCEA1 knockdown in myeloid leukemia cell lines

  • Assess effects on proliferation, differentiation, apoptosis, and clonogenic potential

  • Monitor expression of early neutrophil genes (Mpo, Prtn3, Elane) and differentiation markers (Ltf, Mmp9, Crisp3)

  • Analyze key transcription factors associated with leukemogenesis (C/EBPα, C/EBPε, GFI-1, IRF8)

• Genomic stability assessment:

  • Quantify DNA damage markers (γH2AX, 53BP1) in TCEA1-depleted leukemic cells

  • Measure R-loop formation using S9.6 antibody

  • Perform RNase H rescue experiments to establish causality

• Therapeutic targeting potential:

  • Test whether restoring TCEA1 function affects leukemic cell properties

  • Investigate synthetic lethality approaches targeting pathways dependent on TCEA1 status

Research has shown that downregulation of TCEA1 is observed in certain lymphoma cells compared to germinal center B cells, suggesting a potential relationship between TCEA1 function and hematopoietic malignancies . The effects of TCEA1 knockdown (enhanced proliferation, differentiation blockage, apoptosis inhibition) mirror hallmark features of acute myeloid leukemia, indicating TCEA1 could represent a novel factor in leukemogenesis.

What are the key differences between TCEA1, TCEA2, and TCEA3 antibodies?

Researchers should understand these key differences when selecting antibodies targeting TCEA transcription elongation factors:

Important technical considerations:

• Some commercial antibodies (like EPR14821) recognize all three TCEA family members, making it critical to verify specificity when studying a particular TCEA protein

• For studies specifically targeting TCEA1, researchers should:

  • Confirm antibody specificity using knockout/knockdown controls

  • Use TCEA1-specific primers for mRNA expression validation

  • Consider tissue expression patterns when interpreting results

• When interpreting published literature, note whether the antibody used distinguishes between TCEA family members, as this significantly impacts data interpretation

How can researchers optimize TCEA1 antibody detection in different sample types?

Optimization strategies for TCEA1 antibody detection vary by sample type and application:

Cell Lines:

• HeLa cells: Standard fixation protocols work well; 1:500 dilution for IF, 1:250 for flow cytometry

• HUVEC cells: 4% paraformaldehyde fixation recommended; 1:500 dilution for IF

• 293T cells: Effective for overexpression studies; 1:10000 dilution for Western blot

Tissue Samples:

• Human brain tissue: Antigen retrieval with TE buffer pH 9.0; alternative: citrate buffer pH 6.0

• Human bladder transitional cell carcinoma: 1:500 dilution with hematoxylin counterstain

Sample Preparation Protocols:

• Western Blot:

  • Lysate preparation: RIPA buffer with protease inhibitors

  • Loading: 20 μg protein per lane

  • Blocking: 5% NFDM/TBST

  • Primary antibody: 1:500-1:3000 dilution

  • Detection: HRP-conjugated secondary antibody (1:1000-1:5000)

• Immunoprecipitation:

  • Input: 1.0-3.0 mg of total protein lysate

  • Antibody amount: 0.5-4.0 μg

  • Pre-clearing: Protein A/G beads

  • Incubation: Overnight at 4°C

• Immunofluorescence:

  • Fixation options: -20°C acetone (HeLa) or 4% paraformaldehyde (HUVEC)

  • Primary antibody: 1:500 dilution

  • Secondary antibody: Goat anti-Rabbit IgG (Alexa Fluor® 555) at 1:200

  • Counterstain: DAPI for nuclear visualization

What are common pitfalls when using TCEA1 antibodies and how can researchers overcome them?

Researchers frequently encounter these challenges when working with TCEA1 antibodies:

• Cross-reactivity with other TCEA family members:

  • Problem: Some antibodies (like EPR14821) recognize TCEA1, TCEA2, and TCEA3

  • Solution: Include controls with overexpressed individual TCEA proteins; validate with TCEA1-specific siRNA/shRNA; check tissue expression patterns of TCEA2/3 in your experimental system

• Nuclear localization detection challenges:

  • Problem: TCEA1's predominantly nuclear localization can be difficult to preserve

  • Solution: Use gentle fixation methods; optimize nuclear extraction protocols; employ nuclear/cytoplasmic fractionation for Western blot applications

• Variable detection in different cell types:

  • Problem: TCEA1 detection efficiency varies across cell types

  • Solution: Optimize antibody dilution for each cell type; adjust fixation protocols; consider alternative extraction methods for difficult samples

• Antibody performance in high-throughput applications:

  • Problem: Inconsistent results in ChIP-seq or other genome-wide applications

  • Solution: Validate antibody lot-to-lot consistency; optimize chromatin fragmentation; include spike-in controls for normalization

• Detecting TCEA1 in the context of R-loops:

  • Problem: Traditional fixation may disrupt RNA-DNA hybrids

  • Solution: Use gentler crosslinking methods; optimize sonication conditions; consider native ChIP approaches

How can TCEA1 antibodies be integrated into multi-omics approaches to study transcription regulation?

TCEA1 antibodies can be powerfully integrated into multi-omics research approaches:

• ChIP-seq integration:

  • Pair TCEA1 ChIP-seq with RNA-seq to correlate binding with transcriptional output

  • Integrate with GRO-seq or PRO-seq to assess relationship with nascent transcription

  • Compare TCEA1 binding patterns with RNA Polymerase II occupancy maps

  • Analyze overlap with R-loop mapping (DRIP-seq) to identify susceptible regions

• Proteomics approaches:

  • Immunoprecipitate TCEA1 (0.5-4.0 μg antibody per 1-3 mg lysate) followed by mass spectrometry

  • Perform proximity labeling (BioID or APEX) using TCEA1 as bait

  • Study post-translational modifications of TCEA1 and their functional consequences

  • Apply cross-linking mass spectrometry to map TCEA1 interaction surfaces

• Single-cell applications:

  • Optimize TCEA1 antibodies for CyTOF or CITE-seq

  • Develop TCEA1 proximity ligation assays for single-cell protein-protein interaction studies

  • Apply antibodies in single-cell ChIP approaches

• Spatial biology integration:

  • Use TCEA1 antibodies in multiplex immunofluorescence assays

  • Apply in spatial transcriptomics workflows to correlate localization with gene expression

  • Develop TCEA1 detection for MERFISH or seqFISH applications

These integrated approaches would provide unprecedented insights into TCEA1's multifaceted roles in transcription regulation, cellular differentiation, and genomic stability maintenance.