TCEB1 Antibody

What is TCEB1 and why is it important to study?

TCEB1 (Transcription Elongation Factor B Polypeptide 1), also known as Elongin C, is a 12 kDa protein that functions as a critical subunit of the transcription factor B (SIII) complex. The protein plays dual roles in cellular processes: it regulates transcriptional elongation by RNA polymerase II and mediates protein ubiquitination as part of E3 ubiquitin ligase complexes .

TCEB1 is particularly significant in research because:

• It interacts with the von Hippel-Lindau (VHL) tumor suppressor protein

• Dysregulation has been linked to various cancers and neurodegenerative disorders

• It influences HIF-1α signaling through ubiquitination pathways

• It is overexpressed in certain cancer cell lines, including prostate cancer PC-3 and breast cancer SK-BR-3 cells

What applications are TCEB1 antibodies validated for?

TCEB1 antibodies have been validated for multiple experimental applications, with variation depending on the specific antibody product:

Note that optimal dilutions are antibody-specific and may require titration for each experimental system to achieve optimal results .

How do I choose between polyclonal and monoclonal TCEB1 antibodies?

The choice between polyclonal and monoclonal TCEB1 antibodies depends on your experimental goals:

Polyclonal TCEB1 antibodies:

• Recognize multiple epitopes on the TCEB1 protein

• Often provide stronger signals due to binding multiple sites

• Useful for proteins expressed at low levels

• Examples include rabbit polyclonal antibodies (CAB12515, A46321, 12450-1-AP)

Monoclonal TCEB1 antibodies:

• Recognize a single epitope with high specificity

• Provide consistent lot-to-lot reproducibility

• Reduced background in some applications

• Examples include mouse monoclonal antibody (68164-1-IG)

For initial protein detection and characterization, polyclonal antibodies may offer advantages in signal strength. For highly specific applications or when background is problematic, monoclonal antibodies provide greater consistency .

How should TCEB1 antibodies be stored for optimal performance?

Most TCEB1 antibodies require specific storage conditions to maintain activity:

• Store at -20°C (some formulations at -80°C)

• Avoid repeated freeze-thaw cycles by preparing smaller aliquots

• Most formulations are stable for one year after shipment when properly stored

• Antibody buffers typically contain stabilizers like:

  • PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

  • Or PBS with 0.05% NaN3, 40% Glycerol

Some antibodies (20μl sizes) contain 0.1% BSA as an additional stabilizer. Always check product-specific storage recommendations as they may vary between manufacturers .

What are the optimal experimental conditions for TCEB1 detection in Western blot?

For optimal TCEB1 detection by Western blot:

Sample preparation:

• Use standard protein extraction methods with protease inhibitors

• TCEB1 has an observed molecular weight of 12 kDa

• Positive control samples include HeLa cells, MCF-7 cells, mouse testis tissue, and rat testis tissue

Protocol optimization:

• Primary antibody dilutions: 1:2000-1:12000 (polyclonal) or 1:1000 (monoclonal)

• Use 10-15% SDS-PAGE gels for better resolution of low molecular weight proteins

• Transfer to PVDF or nitrocellulose membranes (0.2 μm pore size recommended for small proteins)

• Blocking: 5% non-fat milk or BSA in TBST

• Secondary antibody selection should match host species (anti-rabbit or anti-mouse IgG)

For validation, positive samples that consistently show TCEB1 expression include Jurkat, PC-3, MCF7, HepG2, mouse liver, mouse lung, mouse testis, and mouse brain tissues .

How can I optimize immunohistochemistry protocols for TCEB1 detection in tissue samples?

For effective TCEB1 detection in tissue samples by IHC:

Tissue preparation and antigen retrieval:

• Formalin-fixed paraffin-embedded (FFPE) sections work well

• Optimal antigen retrieval:

  • Citrate buffer pH 6.0 for FFPE tissue sections

  • Alternatively, TE buffer pH 9.0 has been reported effective

Staining protocol:

• Recommended antibody dilutions: 1:50-1:500

• Positive control tissues: human breast cancer tissue, human ovary tumor tissue

• For validated visualization, use appropriate detection systems (HRP/DAB or fluorescent secondary antibodies)

• Counterstaining with hematoxylin provides nuclear context

Validation image example:
Immunohistochemical analysis of paraffin-embedded Human Lung cancer tissue using TCEB1 antibody at dilution 1/100 has demonstrated specific nuclear staining patterns .

What controls should be included when using TCEB1 antibodies in research?

Rigorous experimental design requires appropriate controls:

Positive controls:

• Cell lines: HeLa, MCF-7, PC-3, RPMI8226, Jurkat, HepG2

• Tissues: Mouse/rat testis, human breast cancer tissue, human ovary tumor tissue

Negative controls:

• Primary antibody omission

• Isotype controls (IgG from same species as primary antibody)

• siRNA knockdown of TCEB1 to confirm specificity

• Blocking peptide competition (if available)

Technical controls:

• Loading controls for Western blot (β-actin, GAPDH)

• Tissue architecture controls for IHC (H&E staining of adjacent sections)

• Nuclear counterstain for IF/ICC experiments to confirm nuclear localization

Including these controls ensures antibody specificity and helps troubleshoot any technical issues that may arise during experiments .

How can TCEB1 antibodies be used to study cancer progression mechanisms?

TCEB1 plays important roles in cancer progression through several mechanisms:

Research approaches using TCEB1 antibodies:

• Evaluate TCEB1 expression patterns across cancer types using IHC/IF

• Examine correlations between TCEB1 levels and HIF-1α signaling

• Investigate TCEB1's role in protein degradation pathways affecting oncogenic proteins

Mechanistic insights:

• TCEB1 overexpression has been observed in prostate cancer (PC-3) and breast cancer (SK-BR-3) cell lines

• TCEB1 may promote cancer cell invasion

• The SPRY4-IT1-TCEB1 axis regulates metastasis in cancer cells through modulation of HIF-1α stability

Research has shown that SPRY4-IT1-mediated suppression of TCEB1 can activate HIF-1α signaling pathways. Knockdown of SPRY4-IT1 in cancer cells decreased HIF-1α and MMP-9 protein expression, whereas TCEB1 inhibition rescued this effect, suggesting a regulatory pathway involving TCEB1 in cancer metastasis .

What are the technical challenges in detecting TCEB1 in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) of TCEB1 presents specific challenges due to its role in protein complexes:

Technical considerations:

• TCEB1's small size (12 kDa) requires optimized gel systems for separation from antibody light chains (~25 kDa)

• As part of multiprotein complexes (with Elongin A/B, VHL, or other partners), buffer conditions need careful optimization

Methodological approaches:

• Use antibodies conjugated to beads (avoiding antibody contamination in eluates)

• Consider specialized IP kits designed for small proteins

• When investigating TCEB1 interactions with specific partners:

  • Target the larger binding partner for IP when possible

  • Use RIP (RNA immunoprecipitation) for studying TCEB1-RNA interactions as demonstrated in SPRY4-IT1 research

For studying TCEB1 interactions with lncRNAs like SPRY4-IT1, researchers have successfully used anti-STAU1 RIP to pull down endogenous lncRNAs and mRNAs associated with STAU1, demonstrating that STAU1 immunoprecipitates were significantly enriched in both SPRY4-IT1 and TCEB1 compared to control IgG .

How do different TCEB1 antibodies perform in detecting specific isoforms or post-translational modifications?

TCEB1 exists in multiple isoforms and can undergo post-translational modifications that affect its function:

Isoform detection:

• Human TCEB1 has a canonical form (112 amino acids) and at least one alternative isoform

• Most commercial antibodies target sequences common to known isoforms

• Antibodies raised against specific regions may detect different isoform subsets:

  • Antibodies targeting the N-terminus (amino acids 1-112) detect the canonical form

  • For isoform-specific detection, check the immunogen sequence information in product datasheets

Post-translational modification detection:

• Standard TCEB1 antibodies detect total protein regardless of modification state

• For phosphorylation or ubiquitination studies, specialized modification-specific antibodies may be required

• When studying TCEB1 in ubiquitination complexes:

  • Use co-immunoprecipitation with denaturing conditions to preserve modifications

  • Consider dual immunostaining approaches to detect modifications in situ

Currently, most research relies on total TCEB1 detection rather than specific modified forms, representing an area for future antibody development .

How can TCEB1 antibodies help elucidate the role of TCEB1 in transcriptional regulation?

TCEB1 (Elongin C) functions in transcriptional regulation as part of the SIII complex:

Experimental approaches:

• Chromatin Immunoprecipitation (ChIP):

  • Use TCEB1 antibodies to identify genomic binding sites

  • Combine with RNA Pol II ChIP to correlate with transcriptional activity

• Immunofluorescence co-localization:

  • Dual staining with TCEB1 and transcription factor antibodies

  • Nuclear localization pattern analysis during active transcription

• Functional studies:

  • Combine TCEB1 knockdown with TCEB1 antibody detection to validate targets

  • Use TCEB1 antibodies to monitor complex formation with Elongin A and B

Research context:
TCEB1 functions as a subunit of the transcription factor B (SIII) complex alongside Elongins A/A2 and B. This complex activates elongation by RNA polymerase II by suppressing transient pausing of the polymerase at many sites within transcription units. While Elongin A functions as the transcriptionally active component, TCEB1 (Elongin C) serves as a regulatory subunit .

What are common issues with TCEB1 antibody specificity and how can they be addressed?

Researchers may encounter specificity issues when using TCEB1 antibodies:

Common specificity problems:

• Cross-reactivity with related SKP1 family proteins

• Non-specific binding due to high antibody concentrations

• Batch-to-batch variation (especially with polyclonal antibodies)

Solutions and verification approaches:

• Validation strategies:

  • Genetic approaches: siRNA/shRNA knockdown of TCEB1

  • Overexpression controls: Compare with TCEB1-overexpressing cells

  • Use multiple antibodies targeting different epitopes

• Optimization techniques:

  • Titrate antibody concentrations to minimize background

  • Add blocking peptides to confirm specificity

  • Increase washing time/stringency to reduce non-specific binding

• Technical considerations:

  • For polyclonal antibodies (e.g., 12450-1-AP), antigen affinity purification improves specificity

  • For monoclonal antibodies (e.g., 68164-1-IG), Protein A purification ensures high purity

Researchers should verify manufacturer's validation data, which typically includes Western blot images showing a single band at the expected 12 kDa size in positive control samples .

How can researchers validate the reproducibility of TCEB1 antibody performance across different experimental batches?

Ensuring reproducible results with TCEB1 antibodies requires systematic validation:

Batch validation protocol:

• Reference sample testing:

  • Maintain frozen aliquots of positive control lysates (e.g., HeLa or MCF-7 cells)

  • Test new antibody batches alongside these standards

  • Compare signal intensity, background, and specificity patterns

• Standardization approaches:

  • Document optimal dilutions for each application

  • Use validated lysate preparation protocols consistently

  • Implement quantitative measurement of signal-to-noise ratios

• Record keeping:

  • Maintain detailed notes on antibody lot numbers

  • Document storage conditions and freeze-thaw cycles

  • Note any variations in experimental conditions

Reproducibility considerations:

• Monoclonal antibodies (e.g., 68164-1-IG) typically show better lot-to-lot consistency

• Polyclonal antibodies may require re-titration with each new lot

• Some manufacturers provide lot-specific validation data upon request

Incorporating these validation steps helps ensure experimental reproducibility and facilitates troubleshooting when performance issues arise.

What factors influence the detection threshold of TCEB1 in different sample types?

Several factors affect TCEB1 detection sensitivity across different experimental samples:

Sample-specific considerations:

• Cell/tissue type variations:

  • Endogenous expression levels vary (high in testis tissue, moderate in cell lines)

  • Complex tissue matrices can increase background or mask signals

  • Fixation methods affect epitope accessibility in IHC/IF applications

• Protein extraction efficiency:

  • Nuclear proteins require specialized extraction methods

  • TCEB1's small size (12 kDa) may lead to loss during some extraction procedures

  • Protein-protein interactions may sequester TCEB1 in complexes

Methodological factors affecting detection threshold:

• Signal amplification systems (enhanced chemiluminescence, tyramide signal amplification)

• Primary antibody concentration and incubation time

• Secondary antibody selection and detection system sensitivity

• For low abundance samples, concentration steps may be required

Optimization table for detecting low levels of TCEB1:

Researchers should systematically test these variables to determine optimal conditions for their specific sample types .

How does TCEB1 function in the regulation of HIF-1α signaling pathways?

TCEB1 plays a crucial role in regulating HIF-1α stability and activity:

Mechanistic pathway:

• TCEB1 (Elongin C) forms part of the E3 ubiquitin ligase complex with VHL protein

• This complex targets HIF-1α for ubiquitination and subsequent proteasomal degradation

• Under normal oxygen conditions, this pathway maintains low HIF-1α levels

• Disruption of TCEB1 function can lead to HIF-1α stabilization and increased activity

Research evidence:
Research has shown that suppression of TCEB1 leads to enhanced HIF-1α and MMP-9 protein expression. In a study on SPRY4-IT1, knockdown of SPRY4-IT1 in cancer cells decreased HIF-1α and MMP-9 protein expression, whereas TCEB1 inhibition rescued this effect .

Specifically, overexpression of HIF-1α in cancer cell lines rescued the diminished migration and invasion ability induced by SPRY4-IT1 knockdown, whereas in other cell lines, the promoting effect of SPRY4-IT1 on migration and invasion was dampened with HIF-1α knockdown. This suggests HIF-1α is a downstream effector of the SPRY4-IT1-TCEB1 axis during cancer metastasis .

What is the significance of TCEB1 in cancer research and potential therapeutic approaches?

TCEB1 has emerged as an important factor in cancer biology:

Cancer relevance:

• Overexpressed in prostate cancer (PC-3) and breast cancer (SK-BR-3) cell lines

• Promotes invasion of prostate cancer cells

• Part of the VHL/HIF pathway frequently dysregulated in clear cell renal cell carcinoma

• Mediates protein degradation of multiple cancer-related proteins

Research applications:

• Biomarker potential: TCEB1 expression correlates with altered HIF-1α signaling

• Higher levels of SPRY4-IT1 correlate with repressed TCEB1 protein levels in human colorectal, breast, and ovary cancer tissues

• Pathway mapping: TCEB1 antibodies help delineate ubiquitination pathways in tumors

Therapeutic implications:

• Targeting the TCEB1-containing E3 ligase complexes may modulate HIF-1α activity

• The SPRY4-IT1-TCEB1-HIF-1α axis represents a potential intervention point

• Monitoring TCEB1 levels could help assess response to HIF pathway inhibitors

Recent research demonstrates that ectopic overexpression of TCEB1 in SPRY4-IT1-overexpressing cancer cells could partially attenuate the increased cell migration and invasion mediated by SPRY4-IT1 overexpression, suggesting TCEB1 as a potential therapeutic target .

How do TCEB1-containing complexes differ across cell types and physiological conditions?

TCEB1 participates in diverse protein complexes with cell type-specific and condition-dependent variations:

Major TCEB1-containing complexes:

• Elongin complex (SIII):

  • Components: Elongin A/A2 + Elongin B + TCEB1 (Elongin C)

  • Function: Regulates RNA polymerase II transcriptional elongation

  • Tissue specificity: Elongin A2 is specifically expressed in testis

• E3 ubiquitin ligase complexes:

  • Components: VHL + Elongin B + TCEB1 + Cullin-2 + Rbx1

  • Function: Targets proteins (including HIF-1α) for degradation

  • Regulation: Oxygen-dependent in the case of HIF-1α targeting

Cell type and condition variations:

• In immune cells, TCEB1 participates in T cell regulation pathways

• Under hypoxic conditions, TCEB1-containing E3 ligase activity toward HIF-1α is reduced

• During cancer progression, altered TCEB1 interactions affect metastatic potential

Experimental approaches to study complex variations:

• Co-immunoprecipitation with TCEB1 antibodies followed by mass spectrometry

• Proximity ligation assays to detect protein-protein interactions in situ

• Differential expression analysis across tissue types and conditions

Understanding these variations is critical for interpreting TCEB1 antibody detection patterns across different experimental systems and physiological states .

What is known about TCEB1 interaction with lncRNAs and its impact on mRNA stability?

Recent research has revealed important TCEB1 interactions with long non-coding RNAs (lncRNAs):

TCEB1-lncRNA interactions:

• lncRNA SPRY4-IT1 has been shown to interact with TCEB1 mRNA through Alu elements

• This interaction is mediated by the RNA-binding protein STAU1

• The binding destabilizes TCEB1 mRNA through Staufen1-mediated mRNA decay (SMD)

Molecular mechanism:

• The Alu element of SPRY4-IT1 base-pairs with TCEB1 mRNA (ΔG values of −175 kcal/mol)

• This RNA-RNA interaction recruits STAU1

• STAU1 binding promotes TCEB1 mRNA degradation

• Reduced TCEB1 protein levels lead to stabilization of HIF-1α

Experimental evidence:
Anti-STAU1 RNA immunoprecipitation (RIP) experiments showed significant enrichment of both SPRY4-IT1 and TCEB1 in STAU1 immunoprecipitates compared to IgG controls. Deletion mapping identified that SPRY4-IT1 mutants lacking the Alu element (1–120 bp) did not interact with STAU1, whereas all other mutants showed comparable binding to wild-type .

This mechanism represents a novel regulatory pathway where lncRNAs like SPRY4-IT1 can modulate TCEB1 expression post-transcriptionally, with downstream effects on HIF-1α signaling and cancer metastasis .

What emerging techniques might enhance TCEB1 detection and functional analysis?

Several cutting-edge approaches show promise for advancing TCEB1 research:

Advanced detection methods:

• Super-resolution microscopy:

  • Techniques like STORM or PALM can visualize TCEB1 complexes below diffraction limit

  • Enables spatial mapping of TCEB1 within nuclear subcompartments

• Proximity labeling approaches:

  • BioID or APEX2 fusion proteins to identify TCEB1 interaction networks

  • TurboID for rapid labeling of transient interactions

• Single-molecule techniques:

  • Single-molecule FISH combined with TCEB1 immunostaining

  • Single-molecule tracking of TCEB1 dynamics in living cells

Functional analysis innovations:

• CRISPR-based approaches:

  • CRISPRi for targeted TCEB1 downregulation

  • CRISPR activation for enhanced expression

  • CRISPR base editors for introducing specific mutations

• Proteomics integration:

  • Combining TCEB1 immunoprecipitation with mass spectrometry

  • Targeted protein quantification using parallel reaction monitoring

These emerging techniques will provide researchers with unprecedented resolution and specificity when studying TCEB1 function across different cellular contexts.

How might TCEB1 antibodies be utilized in developing diagnostic or prognostic markers?

TCEB1 antibodies have potential applications in clinical diagnostics and prognostics:

Diagnostic applications:

• IHC-based tissue profiling to identify altered TCEB1 expression in tumors

• Multiplex immunofluorescence combining TCEB1 with other cancer markers

• Liquid biopsy approaches detecting TCEB1 alterations in circulating tumor cells

Prognostic marker development:

• Correlation studies linking TCEB1 levels with patient outcomes

• Integration with other biomarkers to enhance predictive power

• Automated image analysis of TCEB1 staining patterns to quantify expression

Translational research considerations:

• Standardization of TCEB1 detection protocols for clinical implementation

• Validation across multiple patient cohorts and cancer types

• Correlation with functional outcomes (e.g., hypoxia response, metastatic potential)

Research indicates TCEB1 overexpression in specific cancer types and its association with invasion promotion in prostate cancer cells . The SPRY4-IT1-TCEB1-HIF-1α axis has demonstrated relevance to cancer metastasis , suggesting TCEB1 detection could provide valuable clinical information for patient stratification and treatment selection.