TBRG4 Antibody

What is TBRG4 and where is it localized in cells?

TBRG4 (Transforming Growth Factor Beta Regulator 4) is a mitochondrial protein that plays critical roles in processing mitochondrial RNA precursors and stabilizing specific mature mitochondrial RNA species, including MT-CO1, MT-CO2, MT-CYB, MT-CO3, MT-ND3, MT-ND5, and MT-ATP8/6 . It may also participate in cell cycle progression. Immunofluorescence studies consistently confirm its mitochondrial localization, which is essential for its biological functions .

What are the typical applications for TBRG4 antibodies in research?

TBRG4 antibodies have been validated for multiple experimental applications including:

• Western Blot (WB): Detection of TBRG4 protein in cell and tissue lysates

• Immunohistochemistry (IHC): Visualization of TBRG4 expression in tissue sections

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Examination of subcellular localization

• Immunoprecipitation (IP): Isolation of TBRG4 and associated proteins

• Co-immunoprecipitation (Co-IP): Study of protein-protein interactions

• ELISA: Quantitative detection of TBRG4

• HITS-CLIP: RNA-protein interaction studies

What is the molecular weight of TBRG4 and how does this impact Western blot interpretation?

While the calculated molecular weight of TBRG4 is 71 kDa (631 amino acids), researchers should note that the observed molecular weight in Western blots can appear as either 60 kDa or 71 kDa . This discrepancy should be considered when interpreting results, as it may reflect post-translational modifications, alternative splicing variants, or degradation products. When optimizing Western blot protocols, researchers should run appropriate positive controls such as HepG2 or HeLa cell lysates to establish the expected banding pattern .

What are the optimal conditions for using TBRG4 antibodies in common applications?

For reproducible results across applications, researchers should consider these validated conditions:

It is recommended to titrate these antibodies in each testing system to obtain optimal results, as performance can be sample-dependent .

How should antigen retrieval be optimized for TBRG4 immunohistochemistry?

For optimal IHC staining of TBRG4, antigen retrieval should be performed using TE buffer at pH 9.0. Alternatively, citrate buffer at pH 6.0 may be used, though potentially with reduced efficacy. This step is particularly critical when examining TBRG4 expression in tissue samples like human liver cancer tissue, where proper retrieval ensures accessibility of the epitope for antibody binding . The choice of retrieval method can significantly impact staining intensity and specificity, so preliminary optimization experiments comparing both methods are recommended.

What controls should be included in TBRG4 knockdown experiments?

When designing TBRG4 knockdown experiments, particularly for viral reactivation studies, researchers should include:

• Non-targeting control (NTC) or non-silencing (NS) control shRNAs

• Multiple TBRG4-targeting shRNAs to control for off-target effects

• qPCR validation of knockdown efficiency for each shRNA

• Appropriate positive controls for downstream assays

As demonstrated in published studies, different shRNAs can show variable knockdown efficiencies, with significant consequences for experimental outcomes. For example, in KSHV studies, shTBRG4 #1 showed better knockdown efficiency than shTBRG4 #2, correlating with stronger effects on viral gene expression .

How does TBRG4 regulate gammaherpesvirus latency and reactivation?

TBRG4 functions as a cellular repressor of KSHV and EBV reactivation through regulation of reactive oxygen species (ROS) production. Mechanistically, knockdown of TBRG4 in cells latently infected with these viruses induces viral lytic gene transcription and replication through the following pathway:

• TBRG4 depletion causes mitochondrial stress

• This leads to increased ROS production

• Elevated ROS levels promote viral reactivation from latency

• Treatment with ROS scavengers reverses these effects

These findings suggest that TBRG4 maintains viral latency by modulating mitochondrial function and redox homeostasis. The induction of viral reactivation in TBRG4-depleted cells is further enhanced by treatment with chemical inducers such as TPA and sodium butyrate (NaB) .

What is the association between TBRG4 expression and cancer prognosis?

High TBRG4 expression has been associated with unfavorable clinical outcomes in multiple cancer types. In hepatocellular carcinoma (HCC), TBRG4 expression correlates significantly with:

Additionally, studies have identified elevated TBRG4 expression in head and neck squamous cell carcinoma and other tumor tissues, suggesting its potential role as a prognostic biomarker and therapeutic target .

What approaches can be used to study TBRG4's role in mitochondrial RNA processing?

To investigate TBRG4's function in mitochondrial RNA processing, researchers can employ several sophisticated approaches:

• RNA-protein interaction studies: HITS-CLIP or similar techniques to identify direct RNA targets of TBRG4

• Mitochondrial RNA stability assays: Following TBRG4 knockdown/overexpression, measure half-lives of specific mitochondrial transcripts (MT-CO1, MT-CO2, MT-CYB, etc.)

• RNA-seq of mitochondrial transcriptome: Analyze global changes in mitochondrial RNA processing and abundance

• Protein-protein interaction studies: Co-IP followed by mass spectrometry to identify TBRG4 binding partners in the mitochondrial RNA processing machinery

• Mitochondrial function assays: Measure oxidative phosphorylation, membrane potential, and ROS production in TBRG4-manipulated cells

These approaches would help elucidate the molecular mechanisms through which TBRG4 influences the processing and stability of specific mitochondrial RNA species .

How can researchers address non-specific binding in TBRG4 Western blots?

When encountering non-specific binding in TBRG4 Western blots, consider implementing these techniques:

• Optimization of blocking conditions: Use 5% non-fat dry milk or BSA in TBST buffer for 1-2 hours at room temperature

• Antibody titration: Test multiple dilutions (1:500, 1:1000, 1:2000, 1:3000) to determine optimal signal-to-noise ratio

• Include appropriate controls: Run TBRG4 knockdown samples alongside wild-type samples

• Lysate preparation: Ensure complete extraction of mitochondrial proteins using appropriate lysis buffers

• Secondary antibody optimization: Test different secondary antibodies and dilutions

• Membrane washing: Increase washing time and volume of TBST between antibody incubations

If non-specific bands persist, validate their identity by comparing the pattern with published literature showing TBRG4 to typically appear at 60-71 kDa .

What are the key considerations when interpreting TBRG4 immunofluorescence results?

When analyzing TBRG4 immunofluorescence data, researchers should consider:

• Mitochondrial co-localization: Always include a mitochondrial marker (e.g., MitoTracker) to confirm the expected mitochondrial localization of TBRG4

• Signal specificity: Include TBRG4 knockdown controls to verify antibody specificity

• Cell type variations: TBRG4 expression and localization may vary between cell types (e.g., HeLa vs. U2OS)

• Fixation method impact: Different fixation methods (paraformaldehyde vs. methanol) may affect epitope accessibility

• Permeabilization optimization: Mitochondrial proteins may require stronger permeabilization (e.g., 0.2% Triton X-100)

• Signal-to-background ratio: Adjust antibody concentration to optimize specific signal while minimizing background

Published immunofluorescence images show TBRG4 exhibiting a characteristic punctate mitochondrial staining pattern, which should be used as a reference when validating experimental results .

How should researchers interpret contradictory findings regarding TBRG4 function in different experimental systems?

When encountering contradictory results about TBRG4 function across experimental systems:

• Consider cell type-specific effects: TBRG4's role may vary between different cell types due to differences in mitochondrial biology or interacting partners

• Evaluate knockout/knockdown efficiency: Partial vs. complete depletion of TBRG4 may yield different phenotypes

• Examine acute vs. chronic TBRG4 depletion: Compensatory mechanisms may emerge during long-term TBRG4 deficiency

• Assess experimental conditions: Variations in culture conditions, stress levels, or metabolic state can influence TBRG4 function

• Evaluate redundancy with related proteins: Other mitochondrial RNA-binding proteins may compensate for TBRG4 loss in some contexts

• Consider technical limitations: Different antibodies or detection methods may recognize distinct TBRG4 isoforms or modified forms

To resolve contradictions, comprehensive approaches combining multiple techniques (genomic, proteomic, functional) across diverse experimental systems are recommended .

What are emerging applications of TBRG4 research in therapeutic development?

Based on recent findings linking TBRG4 to disease mechanisms, several promising therapeutic directions emerge:

• Viral latency modulation: Given TBRG4's role in maintaining KSHV and EBV latency, targeting this pathway might enable novel strategies for treating virus-associated malignancies through controlled viral reactivation followed by antiviral therapy

• Cancer therapy: The association between high TBRG4 expression and poor cancer outcomes suggests it could serve as both a prognostic biomarker and therapeutic target, particularly in hepatocellular carcinoma

• Mitochondrial disease: TBRG4's function in mitochondrial RNA processing indicates potential relevance to mitochondrial disorders, which could be addressed through RNA-targeted therapies

• ROS-mediated signaling: The connection between TBRG4, mitochondrial function, and ROS generation provides opportunities to develop interventions targeting redox homeostasis in various pathological contexts

Each of these approaches would benefit from further mechanistic studies using TBRG4 antibodies for target validation and pathway elucidation.

How might single-cell analysis enhance our understanding of TBRG4 function?

Single-cell approaches offer unprecedented opportunities to investigate TBRG4 biology:

• Heterogeneity in expression: Single-cell RNA-seq could reveal cell-to-cell variation in TBRG4 expression within tissues and correlate this with mitochondrial gene expression patterns

• Subcellular dynamics: Super-resolution microscopy using TBRG4 antibodies could map its precise distribution within mitochondrial subcompartments and how this changes under different conditions

• Temporal regulation: Live-cell imaging with tagged TBRG4 could track its dynamics during cellular processes like cell division or stress responses

• Functional clustering: Single-cell multi-omics approaches could link TBRG4 expression levels to mitochondrial function, metabolic state, and cellular phenotypes

• Disease relevance: Single-cell analysis of patient samples could identify specific cell populations where TBRG4 dysregulation contributes to pathogenesis