THOC5 Antibody, HRP conjugated
Description
Introduction to THOC5 Antibody, HRP Conjugated
The THOC5 Antibody, HRP conjugated is a specialized immunological reagent designed to target the THOC5 protein, a critical component of the THO (Transcription-Export) complex. This antibody is enzymatically conjugated with Horseradish Peroxidase (HRP), enabling direct detection of THOC5 in Western blotting (WB) without requiring secondary antibodies. It is primarily used to study mRNA processing, transcription elongation, and nuclear export mechanisms, as well as THOC5’s role in stem cell maintenance, cancer progression, and radioresistance .
Tested Applications
The THOC5 antibody is validated for:
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Western Blotting (WB): Detects a 79 kDa band corresponding to THOC5 in HEK-293, HeLa, and NIH/3T3 cells .
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Immunoprecipitation (IP): Efficiently pulls down THOC5 in HEK-293 cells, enabling interaction studies (e.g., with CDK12, DDX5, DDX17) .
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Immunohistochemistry (IHC): Stains THOC5 in human breast and lung cancer tissues, with antigen retrieval via TE buffer (pH 9.0) .
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Immunofluorescence (IF): Visualizes nuclear localization in HepG2 cells .
Recommended Dilutions
| Application | Dilution |
|---|---|
| WB | 1:3000–1:8000 |
| IP | 0.5–4.0 µg/mg lysate |
| IHC | 1:500–1:2000 |
| IF | 1:50–1:500 |
Source: Proteintech (14862-1-AP)
Role in Transcription and mRNA Processing
THOC5 regulates transcription elongation by interacting with CDK12 and RNA helicases (DDX5, DDX17) in slow RNA polymerase II (Pol II) cells. Depletion of THOC5 delays elongation, accumulates Pol II near transcription start sites (TSS), and disrupts alternative polyadenylation .
Stem Cell Maintenance and Cancer Progression
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Radioresistance in Triple-Negative Breast Cancer (TNBC): THOC5 is upregulated in radioresistant TNBC cells and correlates with poor prognosis. Knockdown reduces stemness markers (NANOG, SOX2) and tumor growth in vivo .
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Osteoclastogenesis: THOC5 shuttles between nucleus and cytoplasm during M-CSF signaling, interacting with c-FMS fragments to regulate osteoclast differentiation .
Western Blotting Protocol
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Sample Preparation: Lyse cells in RIPA buffer with protease inhibitors.
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Gel Electrophoresis: Resolve 20–50 µg total protein on SDS-PAGE.
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Transfer: Transfer to PVDF membrane.
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Detection: Incubate with THOC5 antibody (1:3000–1:8000) for 1–2 hours. Use HRP-conjugated secondary if unconjugated primary is used .
Optimization Tips
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Antigen Retrieval: For IHC, use TE buffer (pH 9.0) or citrate buffer (pH 6.0) .
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Blocking: Use 5% BSA for IP to reduce non-specific binding .
Clinical and Therapeutic Implications
THOC5’s dual role in transcription regulation and stem cell maintenance positions it as a therapeutic target in cancers with high THOC5 expression, such as TNBC. Its interaction with CDK12 and RNA helicases suggests potential for targeting R-loop formation or transcription elongation to mitigate radioresistance . In osteoclastogenesis, THOC5 inhibition may modulate bone resorption, offering avenues for treating bone-related disorders .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
THOC5 is a component of the THO subcomplex within the TREX complex. This complex is believed to link mRNA transcription, processing, and nuclear export, specifically associating with spliced mRNA, not unspliced pre-mRNA. TREX recruitment to spliced mRNAs is transcription-independent, binding upstream of the exon-junction complex (EJC). Its recruitment is splicing- and cap-dependent, occurring near the 5' mRNA end. Here, it facilitates mRNA export to the cytoplasm via the TAP/NFX1 pathway. The TREX complex is crucial for exporting Kaposi's sarcoma-associated herpesvirus (KSHV) intronless mRNAs and for infectious virus production. THOC5, in conjunction with ALYREF/THOC4, participates in NXF1-NXT1 mediated nuclear export of HSP70 mRNA; both proteins enhance NXF1's RNA binding and are necessary for NXF1's nuclear rim localization. THOC5 is involved in transcription elongation, genome stability, and alternative polyadenylation site selection by recruiting CPSF6 to the 5' region of target genes, likely mediating the interaction between TREX and CFIm complexes. It also regulates the expression of myeloid transcription factors CEBPA, CEBPB, and GAB2 by increasing phosphatidylinositol 3,4,5-trisphosphate levels. THOC5 may contribute to granulocyte and adipocyte differentiation. It's essential for hematopoietic primitive cell survival and plays a key role in monocytic development.
Further Research Highlights:
- Suppression of multiple THOC5 target genes may offer a novel therapeutic strategy for hepatocellular carcinoma (HCC). PMID: 26549021
- THOC5 is implicated in regulating serum HDL-C levels. PMID: 24023261
- THOC5 controls polyadenylation site selection through co-transcriptional loading of CFIm68 onto target genes. PMID: 23685434
- THOC5 Y225 phosphorylation regulates mRNA binding; CXCL12 induces this phosphorylation, modulating motile response. PMID: 23032722
- DNA damage reduces the cytoplasmic pool of THOC5-dependent mRNAs and impairs THOC5/mRNA complex formation. PMID: 21937706
- THOC5 protein expression enhances receptor signaling to transcription factor expression and monocyte differentiation. PMID: 19015024
- THOC7 (amino acids 50-137) directly binds to the N-terminal portion (1-199) of FMIP. PMID: 19059247
- Thoc5 exhibits in vitro RNA-binding activity and is associated with HSP70 mRNPs in vivo as a THO complex component. PMID: 19165146
Q&A
What is the role of THOC5 in cellular biology, and how does it relate to transcription elongation?
THOC5 is a component of the THO subcomplex within the TREX complex, which plays a critical role in coupling mRNA transcription, processing, and nuclear export. Specifically, THOC5 is involved in transcription elongation by interacting with RNA Polymerase II and other factors such as CDK12. This interaction facilitates the recruitment of transcriptional machinery to R-loops and enhances elongation efficiency in a gene-specific manner .
In experimental settings, THOC5 has been shown to regulate alternative polyadenylation site choice by recruiting CPSF6 to target genes. This recruitment mediates the association between the TREX complex and cleavage/polyadenylation factors, ensuring proper mRNA processing and export . Furthermore, depletion of THOC5 leads to significant transcriptional delays due to RNA Polymerase II accumulation near transcription start sites (TSS), highlighting its essential role in elongation dynamics .
How can researchers optimize experimental conditions when using THOC5 Antibody conjugated with HRP?
To achieve optimal results with THOC5 Antibody conjugated with HRP, several factors must be considered:
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Dilution Ratios: The recommended dilutions vary depending on the application. For example:
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Antigen Retrieval: For IHC applications, antigen retrieval can significantly impact antibody binding. Suggested buffers include TE buffer at pH 9.0 or citrate buffer at pH 6.0 .
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Storage Conditions: The antibody should be stored at -20°C to maintain stability for up to one year. Avoid repeated freeze-thaw cycles as this can degrade the HRP conjugate .
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Validation: It is crucial to validate the antibody in your specific system using positive controls such as HEK-293 or HeLa cells for WB or IHC applications .
By carefully optimizing these parameters, researchers can ensure reproducibility and reliability in their experiments.
What are the known protein interactions of THOC5, and how do these interactions influence its function?
THOC5 interacts with several key proteins that modulate its functions in transcription and mRNA processing:
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CDK12: This kinase is recruited by THOC5 to R-loops in slow RNA Polymerase II cells, promoting transcription elongation .
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DDX5 and DDX17: These RNA helicases form complexes with THOC5 to regulate RNA processing and stability .
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THOC6: A member of the same complex, THOC6 interacts with THOC5 to facilitate chromatin association and transcription regulation .
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NXF1-NXT1 Complex: In conjunction with ALYREF/THOC4, THOC5 enhances the RNA-binding activity of NXF1 and mediates nuclear export of specific mRNAs such as HSP70 .
These interactions underscore the multifunctional nature of THOC5 in coordinating transcriptional elongation, mRNA processing, and nuclear export.
How does THOC5 depletion affect gene expression at a genome-wide level?
THOC5 depletion has profound effects on gene expression:
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Transcriptional Delays: Depletion causes accumulation of RNA Polymerase II near TSSs of dependent genes, leading to delayed elongation rates .
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Alternative Polyadenylation: Loss of THOC5 disrupts alternative cleavage at polyadenylation sites, affecting mRNA stability and export efficiency .
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Apoptosis Induction: In stem cells, complete knockdown of THOC5 triggers rapid apoptosis due to its essential role in survival pathways .
Genome-wide studies using techniques like DRB/TT-seq have revealed that THOC5 preferentially regulates inducible genes associated with slow RNA Polymerase II dynamics. These findings highlight its critical role in maintaining cellular homeostasis under various conditions.
What are the methodological considerations for studying THOC5-dependent transcription using HRP-conjugated antibodies?
When investigating THOC5-dependent transcription:
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Sample Preparation:
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Detection Techniques:
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For chromatin immunoprecipitation (ChIP), ensure crosslinking efficiency to capture chromatin-associated complexes.
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Use HRP-conjugated antibodies for enhanced signal detection in Western blotting or ELISA assays.
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Controls:
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Include knockdown or knockout controls using shRNA or CRISPR-Cas9 targeting THOC5.
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Validate specificity using recombinant proteins or peptide competition assays.
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These methodological considerations enable precise analysis of THOC5 function at both molecular and cellular levels.
How does HRP conjugation enhance the utility of THOC5 antibodies in experimental workflows?
HRP conjugation offers several advantages:
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Signal Amplification: HRP catalyzes chromogenic or chemiluminescent reactions, producing high-intensity signals that improve sensitivity.
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Versatility: Compatible with multiple detection platforms including ELISA, Western blotting, and immunohistochemistry.
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Quantitative Analysis: Enables accurate quantification of protein levels through densitometry or spectrophotometry.
What challenges might arise when interpreting data from experiments involving THOC5 Antibody?
Several challenges may complicate data interpretation:
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Off-target Effects: Polyclonal antibodies may recognize non-specific epitopes; validation using knockdown/knockout models is essential.
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Batch Variability: Differences between antibody lots can affect reproducibility; always test new batches against established controls.
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Biological Complexity: As a multifunctional protein involved in diverse processes like transcription elongation and mRNA export, isolating specific effects of THOC5 can be challenging.
Addressing these challenges requires rigorous experimental design and thorough validation protocols.
Are there specific cell types or tissues where THOC5 expression is particularly critical?
THOC5 is ubiquitously expressed but exhibits tissue-specific importance:
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Stem Cells: Essential for survival due to its role in maintaining genomic stability and regulating apoptosis pathways .
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Hematopoietic Cells: Regulates expression of myeloid transcription factors like CEBPA and CEBPB during differentiation .
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Cancer Tissues: Elevated expression has been observed in certain cancers; its role in transcription elongation may contribute to tumorigenesis .
These findings suggest that studying THOC5 in context-specific models can provide insights into its physiological and pathological roles.
