TCEB3 Antibody

What is TCEB3 and what is its primary function in cellular processes?

TCEB3, also known as Elongin A, functions as a general transcription elongation factor that enhances RNA polymerase II activity . It forms part of the SIII complex which is involved in increasing the rate of RNA polymerase II-catalyzed transcriptional elongation. The protein is approximately 110 kDa in size and plays a crucial role in regulating gene expression by facilitating efficient transcription elongation. Recent research has demonstrated that TCEB3 may have additional functions beyond its canonical role in transcription, including potential involvement in cancer progression pathways . Understanding TCEB3's multifaceted roles requires specific detection methods, with antibodies being among the most valuable tools for this purpose.

What applications are TCEB3 antibodies commonly used for in research settings?

TCEB3 antibodies have versatile applications in molecular and cellular biology research. Based on available literature, these antibodies are frequently employed in:

• Western Blotting (WB): For detecting and quantifying TCEB3 protein expression in cell and tissue lysates

• Immunofluorescence (IF): For visualizing subcellular localization of TCEB3

• Immunohistochemistry (IHC): For examining TCEB3 expression patterns in tissue sections

• Immunocytochemistry (ICC): For detecting TCEB3 in cultured cells

• ELISA: For quantitative measurement of TCEB3 in various biological samples

• Immunoprecipitation (IP): For isolating TCEB3 protein complexes to study interaction partners

Research has shown that antibody selection should be application-specific, as performance can vary considerably depending on the experimental context. For instance, certain antibody clones may perform optimally in Western blot analysis but yield suboptimal results in immunohistochemistry applications.

How should researchers validate the specificity of TCEB3 antibodies?

Antibody validation is essential for ensuring reliable experimental results. For TCEB3 antibodies, researchers should implement a multi-faceted validation strategy:

• Positive and negative controls: Use tissues or cell lines with known TCEB3 expression levels. For instance, cervical cancer cells (SiHa, HT-3, Hela, C33A) have been shown to express higher levels of TCEB3 compared to normal cells (HaCaT) .

• Knockdown or knockout validation: Perform siRNA-mediated knockdown of TCEB3 (as demonstrated in cervical cancer research) and confirm reduced signal with the antibody .

• Molecular weight verification: Confirm that the detected band in Western blot corresponds to the expected molecular weight of TCEB3 (approximately 110 kDa).

• Cross-reactivity assessment: Test the antibody against related proteins or in species other than the intended target to ensure specificity.

• Epitope analysis: Consider the antibody's target region when interpreting results, especially when studying splice variants or post-translationally modified forms of TCEB3.

How can TCEB3 antibodies be utilized to investigate the role of TCEB3 in cancer progression?

Recent studies have implicated TCEB3 in cancer development, particularly in cervical cancer. TCEB3 antibodies serve as valuable tools for investigating its oncogenic roles through various methodological approaches:

• Expression analysis: Immunohistochemistry with TCEB3 antibodies can reveal upregulation in cancer tissues compared to adjacent normal tissues, as demonstrated in cervical cancer where TCEB3 showed significantly higher expression .

• Functional studies: Following manipulation of TCEB3 expression (e.g., siRNA knockdown), researchers can use TCEB3 antibodies to confirm expression changes at the protein level before assessing functional outcomes such as proliferation, invasion, and apoptosis .

• Mechanistic investigations: TCEB3 antibodies can help elucidate regulatory mechanisms involving TCEB3. For example, studies have shown that TCEB3 is regulated by the circ-0000212/miR-140-3p axis in cervical cancer, affecting downstream pathways .

• Biomarker potential assessment: Analysis of TCEB3 expression using specific antibodies can help evaluate its correlation with clinical outcomes. Research has shown that high TCEB3 expression correlates with lower survival rates in cervical cancer patients .

• Therapeutic target validation: TCEB3 antibodies can be used to monitor changes in expression following experimental therapeutic interventions, helping to establish TCEB3 as a potential treatment target.

What considerations are important when using TCEB3 antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) experiments using TCEB3 antibodies require careful planning:

• Antibody selection: Choose antibodies specifically validated for immunoprecipitation applications. Not all TCEB3 antibodies work effectively for Co-IP, even if they perform well in other applications.

• Epitope accessibility: Consider whether the antibody's target epitope might be masked by protein-protein interactions. N-terminal antibodies (such as ABIN6261518) may be advantageous for capturing TCEB3 complexes if the N-terminus remains accessible in vivo .

• Cross-linking considerations: Determine whether chemical cross-linking is necessary to stabilize transient interactions between TCEB3 and its binding partners.

• Buffer optimization: TCEB3 interactions may be sensitive to buffer conditions. Optimize salt concentration, detergent type/concentration, and pH to maintain physiologically relevant interactions while minimizing non-specific binding.

• Controls: Include appropriate controls such as IgG controls, input samples, and when possible, TCEB3-depleted samples (e.g., from siRNA knockdown experiments) to confirm specificity.

• Validation of interactions: Confirm suspected interactions through reciprocal Co-IP or alternative methods such as proximity ligation assays or mass spectrometry.

How can researchers investigate the relationship between TCEB3 and microRNA regulatory networks using TCEB3 antibodies?

Research has identified important regulatory relationships between TCEB3 and microRNAs, particularly miR-140-3p in cervical cancer. To investigate these relationships, researchers can employ TCEB3 antibodies in multi-faceted approaches:

• Expression correlation analysis: Use TCEB3 antibodies in Western blot or IHC to assess protein expression levels alongside qRT-PCR for miRNA quantification, enabling correlation analysis between TCEB3 protein levels and miRNA expression .

• Functional studies with miRNA manipulation: Following miRNA mimic or inhibitor transfection, use TCEB3 antibodies to quantify resulting changes in TCEB3 protein expression. For example, research has shown that miR-140-3p inhibitors increase TCEB3 expression in cervical cancer cells .

• Dual-validation approaches: Combine luciferase reporter assays to confirm direct miRNA-mRNA interactions with antibody-based protein detection to verify the functional impact on protein expression .

• Downstream pathway analysis: After establishing miRNA-TCEB3 relationships, use TCEB3 antibodies alongside antibodies against downstream markers (e.g., Ki-67, MMP-2, MMP-9) to map the complete signaling pathway affected by this regulatory axis .

• Circular RNA interactions: When investigating complex regulatory networks involving circular RNAs (like circ-0000212), TCEB3 antibodies can help validate the final protein-level outcomes in the proposed regulatory cascade .

What is the recommended protocol for using TCEB3 antibodies in Western blot analysis?

For optimal Western blot results with TCEB3 antibodies, researchers should follow this methodological approach:

• Sample preparation:

  • Lyse cells or tissues in RIPA buffer containing protease inhibitors

  • Sonicate briefly to shear DNA and reduce sample viscosity

  • Centrifuge at 14,000×g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• Gel electrophoresis:

  • Use 8% SDS-PAGE gels (due to TCEB3's large size of 110 kDa)

  • Load 20-40 μg of total protein per lane

  • Include molecular weight markers

• Transfer:

  • Transfer proteins to PVDF membrane (preferable for large proteins)

  • Use wet transfer system at 100V for 2 hours or overnight at 30V (4°C)

• Blocking:

  • Block membrane with 5% non-fat milk in TBST for 1-2 hours at room temperature

• Primary antibody incubation:

  • Dilute TCEB3 antibody according to manufacturer's recommendation (e.g., 1:1000-1:2000)

  • Incubate overnight at 4°C with gentle agitation

• Washing:

  • Wash membranes 3-5 times with TBST, 5 minutes each

• Secondary antibody:

  • Incubate with appropriate HRP-conjugated secondary antibody

  • Typically use 1:5000-1:10000 dilution for 1 hour at room temperature

• Detection:

  • Develop using ECL substrate and image using digital imaging system

  • Expected band size for TCEB3 is approximately 110 kDa

• Controls:

  • Include loading control (e.g., β-actin, GAPDH)

  • Consider positive control (cell line with known TCEB3 expression)

  • When possible, include a TCEB3 knockdown sample as negative control

What are the optimal conditions for TCEB3 detection using immunofluorescence?

For successful immunofluorescence experiments with TCEB3 antibodies, follow this methodological protocol:

• Cell preparation:

  • Culture cells on coverslips or chamber slides

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Blocking:

  • Block with 5% normal serum (matching the species of secondary antibody) in PBS for 1 hour

• Primary antibody:

  • Dilute TCEB3 antibody to appropriate concentration (typically 1:100-1:500)

  • Incubate overnight at 4°C in a humidified chamber

• Washing:

  • Wash 3 times with PBS, 5 minutes each

• Secondary antibody:

  • Use fluorochrome-conjugated secondary antibody (1:200-1:1000 dilution)

  • Incubate for 1 hour at room temperature in the dark

• Nuclear counterstain:

  • Stain with DAPI (1:1000) for 5 minutes

• Mounting:

  • Mount slides using anti-fade mounting medium

• Imaging:

  • Image using confocal or fluorescence microscopy

  • TCEB3 typically shows nuclear localization with possible cytoplasmic staining

• Controls:

  • Include secondary-only control

  • Consider TCEB3 silencing control (siRNA treated cells)

How should researchers design and validate TCEB3 knockdown experiments using TCEB3 antibodies?

TCEB3 knockdown experiments have proven valuable in understanding its biological functions, as demonstrated in cervical cancer research. A comprehensive validation approach includes:

• siRNA design:

  • Design 2-3 different siRNA sequences targeting different regions of TCEB3 mRNA

  • Include non-targeting scrambled siRNA as negative control

• Transfection optimization:

  • Optimize transfection conditions for each cell line

  • Test different siRNA concentrations (typically 10-50 nM)

  • Determine optimal post-transfection time point for analysis (48-72 hours)

• Knockdown validation at mRNA level:

  • Extract RNA and perform qRT-PCR for TCEB3

  • Normalize to reference genes (e.g., GAPDH, β-actin)

  • Calculate fold change compared to control

• Protein-level validation using TCEB3 antibodies:

  • Perform Western blot analysis using specific TCEB3 antibodies

  • Quantify band intensity and normalize to loading control

  • Aim for at least 70% reduction in protein level

• Functional experiments:

  • Proceed with functional assays only after confirming knockdown

  • Include assays relevant to TCEB3 function (e.g., proliferation, invasion, apoptosis assays)

  • Monitor expression of downstream targets (Ki-67, MMP-2, MMP-9) using specific antibodies

• Documentation:

  • Report both mRNA and protein knockdown efficiency

  • Include representative Western blot images showing TCEB3 reduction

Research has shown that effective TCEB3 knockdown inhibits cell proliferation and invasion while promoting apoptosis in cervical cancer cells, demonstrating its oncogenic potential .

What are the critical factors for successful quantification of TCEB3 using ELISA?

ELISA provides a sensitive method for quantitative measurement of TCEB3 in various biological samples. For optimal results with TCEB3 ELISA:

• Sample preparation:

  • Process samples according to type (serum, plasma, tissue homogenates, cell culture supernatants)

  • For tissue samples, thoroughly homogenize in appropriate buffer

  • Centrifuge samples to remove particulates

  • Consider dilution series to ensure readings fall within the standard curve

• Standards preparation:

  • Prepare fresh standards for each assay

  • Ensure accurate serial dilution

  • The typical detection range for TCEB3 ELISA is 0.32-20 ng/mL

• Assay procedure:

  • Follow sandwich ELISA protocol as specified by the manufacturer

  • Adhere to specified incubation times and temperatures (typically 37°C)

  • Perform multiple washing steps thoroughly to reduce background

• Detection and analysis:

  • Read optical density at 450nm with correction at 570nm or 630nm

  • Generate standard curve using four-parameter logistic curve-fit

  • Calculate sample concentrations from the standard curve

  • Account for any dilution factors in final concentration calculations

• Quality control:

  • Run samples in duplicate or triplicate

  • Include quality control samples of known concentration

  • Verify minimal detectable dose (typically <0.156 ng/mL for TCEB3)

• Specificity considerations:

  • Be aware of potential cross-reactivity with TCEB3 analogues

  • Validate results with alternative methods when possible

How does the sensitivity and specificity of ELISA compare with other methods for TCEB3 detection?

When selecting detection methods for TCEB3, researchers should consider the comparative advantages of different techniques:

• Sensitivity comparison:

  • ELISA: High sensitivity with detection limits typically less than 0.156 ng/mL

  • Western blot: Moderate sensitivity, generally requires higher protein concentrations

  • Immunohistochemistry: Moderate sensitivity, but provides spatial information

  • Mass spectrometry: Variable sensitivity depending on instrument and sample preparation

• Specificity comparison:

  • ELISA: High specificity with properly validated antibody pairs, though cross-reactivity may occur

  • Western blot: Provides specificity verification through molecular weight confirmation

  • Immunoprecipitation-mass spectrometry: Highest specificity for confirming protein identity

• Quantification accuracy:

  • ELISA: Most accurate for absolute quantification within the detection range

  • Western blot: Semi-quantitative, requires careful standardization

  • qPCR (for mRNA): Indirect measure of protein levels, may not correlate perfectly

• Sample requirements:

  • ELISA: Requires intact protein in solution, minimal processing

  • Western blot: Requires denatured protein samples

  • IHC/IF: Requires fixed tissue/cells with preserved epitopes

• Throughput considerations:

  • ELISA: Higher throughput, allows processing multiple samples simultaneously

  • Western blot: Lower throughput, more labor-intensive

  • Automated IHC: Moderate throughput with specialized equipment

The optimal detection method depends on research objectives, available sample types, and required sensitivity/specificity balance. For precise quantification of TCEB3 across multiple samples, ELISA offers significant advantages, while Western blot provides better specificity validation and molecular weight confirmation.

What are common issues encountered when using TCEB3 antibodies and how can they be resolved?

Researchers working with TCEB3 antibodies may encounter several technical challenges. Here are methodological solutions to common problems:

• Low or no signal in Western blot:

  • Increase antibody concentration or extend incubation time

  • Optimize protein loading (try 40-60 μg per lane for TCEB3)

  • Use enhanced chemiluminescence detection systems

  • Consider extracting nuclear fraction for enrichment (as TCEB3 is primarily nuclear)

  • Verify sample preparation method preserves TCEB3 integrity

• Multiple bands or unexpected band sizes:

  • Confirm antibody specificity using knockdown controls

  • Consider potential splice variants or post-translational modifications

  • Use gradient gels for better resolution of high molecular weight proteins

  • Increase washing stringency to reduce non-specific binding

  • Verify the epitope region recognized by the antibody

• High background in immunostaining:

  • Optimize blocking (try 5% BSA instead of serum)

  • Increase wash frequency and duration

  • Dilute primary antibody further

  • Reduce secondary antibody concentration

  • Include 0.1% Tween-20 in antibody diluent

• Inconsistent ELISA results:

  • Ensure proper plate washing between steps

  • Verify reagent storage conditions and expiration dates

  • Prepare fresh standards for each assay

  • Maintain consistent incubation times and temperatures

  • Pre-clear samples by centrifugation before adding to wells

• Poor reproducibility between experiments:

  • Standardize protocols, particularly incubation times and temperatures

  • Use the same antibody lot when possible

  • Implement more detailed record-keeping of experimental conditions

  • Include consistent positive controls across experiments

How can researchers optimize detection of TCEB3 in different tissue and cell types?

Detecting TCEB3 across diverse biological samples requires optimization strategies tailored to sample type:

• Cell lines:

  • For cancer cell lines with high TCEB3 expression (e.g., SiHa, Hela), standard protocols are usually sufficient

  • For cells with lower expression, increase protein loading or use more sensitive detection methods

  • Consider nuclear extraction to enrich for TCEB3 protein

• Tissue samples:

  • Optimize fixation conditions for IHC (10% neutral buffered formalin for 24-48 hours typically works well)

  • Consider antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • For frozen sections, test different fixatives (4% PFA vs. acetone)

  • Adjust primary antibody concentration based on tissue type

• Challenging sample types:

  • For FFPE tissues, extend antigen retrieval time

  • For mouse tissues using mouse-derived antibodies, use specialized blocking reagents to reduce background

  • For highly autofluorescent tissues, consider Sudan Black B treatment before immunofluorescence

• Sample processing considerations:

  • Process tissues rapidly after collection to prevent protein degradation

  • Use protease inhibitors in all extraction buffers

  • For cell lines, harvest cells at consistent confluence levels

  • Standardize protein extraction methods across experiments

• Application-specific optimization:

  • For Western blot: Consider gradient gels for better resolution of high molecular weight proteins

  • For IHC: Test both DAB and fluorescent detection systems

  • For IF: Try different fixation and permeabilization combinations

Careful optimization and standardization of detection protocols enhance reproducibility and facilitate meaningful comparisons across different experimental conditions and sample types.

How is TCEB3 research evolving, and what new applications for TCEB3 antibodies are emerging?

The field of TCEB3 research is expanding beyond its canonical role in transcription elongation, with several emerging research directions:

• Cancer biology applications:

  • TCEB3 is increasingly recognized as a potential oncogene, particularly in cervical cancer

  • TCEB3 antibodies are being used to evaluate its expression as a potential prognostic biomarker

  • Research shows high TCEB3 expression correlates with lower patient survival rates

• Regulatory network investigations:

  • The discovery of the circ-0000212/miR-140-3p/TCEB3 regulatory axis represents a new paradigm in understanding TCEB3 regulation

  • TCEB3 antibodies are essential for validating these complex regulatory networks at the protein level

• Therapeutic target validation:

  • Silencing TCEB3 inhibits cancer cell proliferation and invasion while promoting apoptosis, suggesting therapeutic potential

  • TCEB3 antibodies will be crucial for validating target engagement in future therapeutic development efforts

• Multi-omics integration:

  • Combining TCEB3 protein detection with transcriptomics and epigenomics data provides comprehensive understanding of its regulatory mechanisms

  • Antibody-based proteomics approaches will enable correlation of TCEB3 expression with global proteome changes

• Technological advances:

  • Development of higher-specificity monoclonal antibodies targeting different TCEB3 epitopes

  • Application of single-cell proteomics techniques using TCEB3 antibodies to understand cell-to-cell variability

  • Integration of TCEB3 antibodies into multiplexed imaging technologies for spatial context

As research continues to uncover new functions of TCEB3 and its involvement in diverse cellular processes, TCEB3 antibodies will remain essential tools for advancing our understanding of this multifaceted protein and its potential as a therapeutic target.