TRIM4 Antibody

What is TRIM4 and what cellular functions does it perform?

TRIM4 is a member of the TRIM/RBCC protein family with a canonical length of 500 amino acid residues and a mass of approximately 57.5 kDa in humans. It contains characteristic domains including RING, B-Box, and coiled-coil motifs, which are essential for its function . TRIM4 is primarily localized in the cytoplasm and is widely expressed across numerous tissue types. This protein plays significant roles in:

• Protein ubiquitination as an E3 ubiquitin-protein ligase

• Regulation of innate immune responses, particularly in antiviral defense mechanisms

• Mediating SET protein degradation through K48-linked polyubiquitination

• Modulating hormonal responses in certain cancer types, particularly breast cancer

TRIM4 is notably involved in catalyzing K48-linked polyubiquitination of specific targets, promoting their proteasomal degradation. This function has significant implications for cellular regulation across multiple pathways .

What are the common applications for TRIM4 antibodies in research?

TRIM4 antibodies serve as valuable tools for investigating TRIM4 protein expression, localization, and function across multiple research applications. The most frequently employed techniques include:

• Western Blot: The most widely utilized application for detecting TRIM4 protein expression levels and molecular weight verification

• Immunohistochemistry (IHC): For examining TRIM4 expression patterns in tissue sections

• Immunofluorescence (IF): For visualizing subcellular localization of TRIM4

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of TRIM4 in various samples

These applications allow researchers to investigate TRIM4's role in diverse cellular processes, particularly in immune regulation and cancer biology contexts .

What sample types can be analyzed using TRIM4 antibodies?

TRIM4 antibodies have demonstrated reactivity against human TRIM4 protein and can be used to analyze various sample types:

• Cell lysates from cultured cell lines (particularly useful in cancer research with cell lines such as MCF7, T47D, MDA-MB-231, and MARC145)

• Tissue sections (through IHC techniques)

• Protein extracts from primary tissue samples

• Organoid cultures (particularly relevant for breast cancer research)

Some TRIM4 antibodies may cross-react with orthologs from other species including mouse, rat, bovine, and chimpanzee models, making them valuable for comparative studies across species. Macaca mulatta TRIM4 has been specifically reported to be genetically similar to human TRIM4, making non-human primates potentially useful models for studying TRIM4-related human conditions .

How can TRIM4 antibodies be used to investigate its role in breast cancer?

TRIM4 antibodies provide critical tools for investigating TRIM4's emerging role in breast cancer, particularly in endocrine therapy resistance. Research methodologies include:

• Expression analysis in patient samples: IHC staining of breast cancer tissue microarrays can reveal correlations between TRIM4 expression and clinical parameters including:

  • Tumor stage and grade

  • Hormone receptor status (particularly ER-α)

  • Treatment response

  • Patient outcomes

• Mechanistic studies in cell models: Using TRIM4 antibodies in combination with techniques like immunoprecipitation to elucidate the molecular mechanisms connecting TRIM4 to hormone receptor signaling.

• Biomarker validation: Employing TRIM4 antibodies to assess whether TRIM4 expression can predict response to tamoxifen or other endocrine therapies.

Research has demonstrated that downregulation of TRIM4 is associated with tamoxifen resistance in breast cancer cells, while low TRIM4 expression correlates with unfavorable prognosis in patients. TRIM4 increases ER-α expression and sensitivity to tamoxifen through a mechanism involving SET protein degradation, suggesting TRIM4 could be a valuable prognostic biomarker for ER-α positive breast cancer patients undergoing endocrine therapy .

What methodologies are recommended for investigating TRIM4's immunoregulatory functions?

TRIM4's role in immune regulation can be investigated through several methodologies utilizing TRIM4 antibodies:

• Viral infection models: Measuring TRIM4 expression changes following viral infection or poly(I:C) treatment using Western blotting and qPCR.

• Signaling pathway analysis: Examining the impact of TRIM4 on immune signaling components:

  • Phosphorylation status of IRF3, IRF7, and NF-κB p65

  • Expression levels of downstream effectors including IFN-alpha, IFN-beta, RIG-I, and MAVS

• Promoter activity assays: Analyzing how TRIM4 affects the activity of immune-relevant promoters such as IFN-beta, NF-κB, and ISRE.

• Viral replication studies: Assessing how TRIM4 modulation impacts viral load in infection models.

Research has shown that TRIM4 expression increases after viral infection (e.g., PRRSV) and poly(I:C) stimulation. TRIM4 overexpression has been demonstrated to inhibit viral replication while enhancing the expression of multiple immune-related genes and the activity of important immune promoters .

How can researchers differentiate between TRIM4 isoforms using antibodies?

Differentiating between the reported three isoforms of TRIM4 requires careful antibody selection and experimental design:

• Epitope mapping: Select antibodies that target regions unique to specific isoforms or antibodies that recognize epitopes common to all isoforms.

• Western blot optimization: Use high-resolution SDS-PAGE to separate closely migrating isoforms, followed by immunodetection with appropriate antibodies.

• Isoform-specific knockdown/knockout controls: Employ siRNA or CRISPR-Cas9 to selectively deplete specific isoforms as positive controls.

• Mass spectrometry validation: Combine immunoprecipitation using anti-TRIM4 antibodies with mass spectrometry to definitively identify which isoforms are present in your experimental system.

When selecting commercial antibodies, researchers should carefully examine the immunogen information to determine which isoforms the antibody will recognize. For example, antibodies generated against amino acids 1-280 of human TRIM4 would detect isoforms sharing this N-terminal region .

What are the optimal conditions for using TRIM4 antibodies in Western blot applications?

For optimal Western blot results with TRIM4 antibodies, consider the following protocol optimizations:

• Sample preparation:

  • Use RIPA or NP-40 lysis buffers containing protease inhibitors

  • Include phosphatase inhibitors if phosphorylation status is relevant

  • Denature samples at 95°C for 5 minutes in Laemmli buffer

• Gel electrophoresis:

  • Use 10% SDS-PAGE for optimal resolution of the 57.5 kDa TRIM4 protein

  • Include positive control samples with known TRIM4 expression (e.g., MCF7 cells)

• Transfer conditions:

  • Semi-dry or wet transfer at 100V for 60-90 minutes

  • Use PVDF membranes for higher protein binding capacity

• Antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour

  • Primary antibody dilution typically 1:500-1:1000, incubate overnight at 4°C

  • Secondary antibody dilution typically 1:5000, incubate for 1 hour at room temperature

• Detection:

  • Enhanced chemiluminescence (ECL) detection systems work well

  • Expect a band at approximately 57.5 kDa for the canonical isoform

Optimization is particularly important when studying TRIM4 in different experimental contexts, such as breast cancer cell lines versus immune cells, as expression levels may vary significantly .

What controls are essential when using TRIM4 antibodies in research?

Proper controls are critical for ensuring the reliability and specificity of results obtained with TRIM4 antibodies:

• Positive controls:

  • Cell lines with known high TRIM4 expression (e.g., luminal breast cancer cell lines like MCF7)

  • Recombinant TRIM4 protein as a reference standard

• Negative controls:

  • TRIM4 knockdown or knockout samples generated using siRNA or CRISPR-Cas9

  • Cell lines with naturally low TRIM4 expression (e.g., certain TNBC cell lines)

  • Secondary antibody-only controls to assess non-specific binding

• Specificity controls:

  • Pre-absorption of the antibody with the immunizing peptide

  • Use of multiple antibodies targeting different epitopes of TRIM4

  • Mass spectrometry validation of immunoprecipitated proteins

• Loading controls:

  • Standard housekeeping proteins (β-actin, GAPDH, tubulin) for Western blotting

  • Tissue-specific controls for IHC applications

These controls help validate experimental findings and ensure that observed effects are specifically attributable to TRIM4 rather than artifacts or non-specific interactions .

How can TRIM4 antibodies be used to investigate protein-protein interactions?

TRIM4 antibodies are valuable tools for investigating protein-protein interactions involving TRIM4, particularly in the context of ubiquitination pathways and immune signaling:

• Co-immunoprecipitation (Co-IP):

  • Use anti-TRIM4 antibodies to pull down TRIM4 complexes

  • Analyze co-precipitated proteins by Western blot or mass spectrometry

  • This approach has successfully identified interactions between TRIM4 and SET protein

• Proximity ligation assay (PLA):

  • Visualize and quantify TRIM4 interactions with candidate proteins in situ

  • Particularly useful for transient or context-dependent interactions

• Chromatin immunoprecipitation (ChIP):

  • Investigate whether TRIM4 associates with chromatin-bound complexes

  • Relevant for exploring TRIM4's potential roles in transcriptional regulation

• Domain mapping experiments:

  • Combine with deletion mutants to identify interaction domains

  • Research has shown that TRIM4-SET interactions are mediated by the RING and B-box domains of TRIM4 and the carboxyl terminus of SET

These approaches have revealed that TRIM4 targets SET, catalyzing its K48-linked polyubiquitination at specific lysine residues (K150 and K172), which promotes SET's proteasomal degradation and dissociation from regulatory proteins including p53 and PP2A .

What are common issues when working with TRIM4 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with TRIM4 antibodies:

• Low signal intensity:

  • Increase antibody concentration or incubation time

  • Use signal enhancement systems (e.g., biotin-streptavidin amplification)

  • Optimize protein extraction protocols to enrich for TRIM4

  • Consider using fresh antibody aliquots

• High background:

  • Increase blocking time or blocking agent concentration

  • Use more stringent washing conditions

  • Decrease secondary antibody concentration

  • Pre-absorb antibody with non-specific proteins

• Multiple bands in Western blot:

  • May represent different isoforms, post-translational modifications, or degradation products

  • Use TRIM4 knockdown controls to identify specific bands

  • Compare with positive control samples

  • Consider the specificity of the antibody against different TRIM4 domains

• Inconsistent results between experiments:

  • Standardize protocols for sample preparation and antibody dilution

  • Use consistent positive controls across experiments

  • Prepare and store antibody aliquots according to manufacturer recommendations

  • Consider batch effects in antibody production

When troubleshooting, systematic adjustment of one variable at a time is recommended while maintaining appropriate controls to ensure accurate interpretation of results .

How can researchers validate the specificity of their TRIM4 antibody?

Validating TRIM4 antibody specificity is crucial for ensuring reliable research results:

• Genetic validation:

  • Test antibody in TRIM4 knockdown or knockout models

  • Use TRIM4 overexpression systems as positive controls

  • Specific signal should decrease with knockdown and increase with overexpression

• Peptide competition assay:

  • Pre-incubate antibody with the immunizing peptide or recombinant TRIM4

  • Specific binding should be blocked, resulting in signal loss

• Multiple antibody approach:

  • Compare results using antibodies targeting different TRIM4 epitopes

  • Consistent patterns across antibodies suggest specific detection

• Cross-reactivity assessment:

  • Test in samples expressing related TRIM family proteins

  • Evaluate species cross-reactivity if working with non-human models

• Immunoprecipitation-mass spectrometry:

  • Perform IP with the TRIM4 antibody followed by mass spectrometry

  • Confirm the presence of TRIM4 peptides in the immunoprecipitate

These validation steps help ensure that experimental observations are specifically attributable to TRIM4 and not to related proteins or non-specific interactions .

How should researchers interpret changes in TRIM4 expression in cancer studies?

Interpreting TRIM4 expression data in cancer studies requires careful consideration of several factors:

• Context-dependent significance:

  • TRIM4 downregulation in tamoxifen-resistant breast cancer cells indicates potential clinical relevance

  • Low TRIM4 expression correlates with poor prognosis in ER-α positive breast cancer patients

  • TRIM4 expression is associated with luminal breast cancer subtypes but not with TNBC

• Correlation with clinical parameters:

  • The table below summarizes key correlations observed in breast cancer studies:

• Mechanistic interpretation:

  • TRIM4 enhances ER-α expression and action through SET degradation

  • TRIM4-mediated SET degradation releases p53 and PP2A, which further promotes ESR1 gene transcription

  • This mechanistic understanding explains why TRIM4 loss contributes to endocrine therapy resistance

• Translational implications:

  • TRIM4 may serve as a prognostic biomarker for endocrine therapy response

  • Targeting the TRIM4-SET axis could potentially restore endocrine sensitivity

What is known about TRIM4's role in viral immunity and how can antibodies help elucidate this function?

TRIM4 plays an important role in antiviral immunity, which can be investigated using TRIM4 antibodies:

• Expression changes during infection:

  • TRIM4 expression increases following viral infection (e.g., PRRSV) or poly(I:C) stimulation

  • This upregulation suggests TRIM4 is part of the host antiviral response

• Impact on antiviral signaling pathways:

  • TRIM4 enhances the expression of key antiviral molecules:

    • Type I interferons (IFN-alpha, IFN-beta)

    • Pattern recognition receptors (RIG-I)

    • Signaling adaptors (MAVS)

    • Transcription factors (IRF3, IRF7)

    • Effector molecules (OAS1, IFIT3, CCL5)

• Promoter activation:

  • TRIM4 increases the activity of critical immune promoters:

    • IFN-beta promoter

    • NF-κB promoter

    • ISRE (Interferon-Stimulated Response Element)

• Signaling pathway activation:

  • TRIM4 enhances phosphorylation of key signaling molecules:

    • Phosphorylated IRF3

    • Phosphorylated IRF7

    • Phosphorylated NF-κB p65

• Functional impact on viral replication:

  • TRIM4 overexpression inhibits viral replication (demonstrated with PRRSV)

  • This confirms TRIM4's role as a host antiviral factor

TRIM4 antibodies can be used to track these changes and interactions via Western blotting, immunoprecipitation, and immunofluorescence techniques, enabling detailed characterization of TRIM4's role in innate immune responses to viral infection .

How can TRIM4 antibodies be used to investigate its E3 ubiquitin ligase activity?

TRIM4's E3 ubiquitin ligase activity is central to its cellular functions and can be investigated using specialized approaches with TRIM4 antibodies:

• Ubiquitination assays:

  • In vitro ubiquitination assays with recombinant TRIM4 and potential substrates

  • In vivo ubiquitination assays detecting ubiquitinated proteins after TRIM4 manipulation

  • Co-immunoprecipitation followed by ubiquitin Western blotting to detect ubiquitinated target proteins

• Substrate identification:

  • Immunoprecipitation with TRIM4 antibodies followed by mass spectrometry

  • Comparison of the ubiquitinome between TRIM4-deficient and wild-type cells

  • Validation of specific substrates through targeted Co-IP experiments

• Domain-function analysis:

  • Use of mutant TRIM4 constructs lacking specific domains (e.g., RING domain)

  • Comparison of wild-type versus mutant TRIM4 in ubiquitination assays

  • Structural studies to understand the molecular basis of substrate recognition

• Linkage-specific ubiquitination:

  • Use of linkage-specific ubiquitin antibodies to determine ubiquitin chain type

  • TRIM4 has been shown to catalyze K48-linked polyubiquitination of SET at specific lysine residues (K150 and K172)

  • K48-linked chains typically target proteins for proteasomal degradation

These approaches have revealed that TRIM4 mediates the ubiquitination of SET protein, promoting its proteasomal degradation. This activity is dependent on the RING and B-box domains of TRIM4, which interact with the carboxyl terminus of SET to facilitate this process .

What emerging applications of TRIM4 antibodies might advance our understanding of disease mechanisms?

Several emerging applications of TRIM4 antibodies could significantly advance disease mechanism research:

• Single-cell analysis:

  • Combining TRIM4 antibodies with single-cell technologies to examine heterogeneity of TRIM4 expression

  • Particularly relevant in tumor microenvironments to understand cellular subpopulations with different treatment responses

• Patient-derived organoids:

  • Using TRIM4 antibodies to characterize expression in 3D organoid models

  • These models more accurately recapitulate in vivo conditions than traditional cell lines

  • Early research has shown that TRIM4 overexpression in breast cancer patient-derived organoids reduced their number and size while increasing sensitivity to tamoxifen

• Companion diagnostics development:

  • Developing standardized IHC protocols for TRIM4 detection in clinical samples

  • Potential application as a predictive biomarker for endocrine therapy response in breast cancer

• Therapeutic target validation:

  • Using TRIM4 antibodies to monitor protein levels during drug treatment

  • Evaluating whether compounds that modulate TRIM4 expression or activity affect disease progression

• Post-translational modification mapping:

  • Investigating how TRIM4 itself is regulated through post-translational modifications

  • Understanding the upstream regulators of TRIM4 expression and activity

These applications could yield new insights into TRIM4's role in disease processes and potentially identify novel therapeutic opportunities, particularly in breast cancer and viral infections .

How might TRIM4 antibodies be used to investigate its potential as a biomarker in personalized medicine?

TRIM4 antibodies have significant potential for advancing personalized medicine applications, particularly in oncology:

• Standardized diagnostic testing:

  • Development of validated IHC protocols for TRIM4 detection in clinical pathology

  • Establishment of scoring systems and cutoff values for "TRIM4-low" versus "TRIM4-high" tumors

• Predictive biomarker validation:

  • Retrospective analysis of archived tumor samples with known treatment outcomes

  • Prospective clinical trials incorporating TRIM4 testing to guide treatment decisions

  • Multivariate analyses to determine TRIM4's independent predictive value

• Combination biomarker panels:

  • Integration of TRIM4 with other markers (e.g., ER-α, PR, HER2) for improved predictive power

  • Development of multiplexed immunofluorescence approaches to simultaneously detect multiple markers

• Liquid biopsy development:

  • Investigation of circulating TRIM4 protein or autoantibodies as non-invasive biomarkers

  • Potential for monitoring treatment response and disease progression

• Theranostic applications:

  • Development of antibody-drug conjugates targeting TRIM4

  • Imaging applications using labeled TRIM4 antibodies

What methodological advances might improve the utility of TRIM4 antibodies in complex experimental systems?

Several methodological advances could enhance the utility of TRIM4 antibodies in complex experimental systems:

• Advanced imaging techniques:

  • Super-resolution microscopy for precise subcellular localization

  • Live-cell imaging with fluorescently tagged anti-TRIM4 nanobodies

  • Expansion microscopy to visualize TRIM4 interactions at the nanoscale

• Multiplexed detection systems:

  • Cyclic immunofluorescence for analyzing multiple proteins in the same sample

  • Mass cytometry (CyTOF) incorporating TRIM4 antibodies for high-dimensional single-cell analysis

  • Spatial transcriptomics combined with TRIM4 IHC to correlate protein expression with gene expression patterns

• High-throughput screening applications:

  • Automated IHC or IF platforms for large-scale tissue analysis

  • Cell-based screens to identify modulators of TRIM4 expression or activity

  • Patient-derived xenograft libraries for in vivo modeling

• Improved antibody engineering:

  • Development of recombinant antibodies with increased specificity

  • Single-domain antibodies (nanobodies) for improved tissue penetration

  • Bispecific antibodies targeting TRIM4 and interaction partners simultaneously

• In situ proximity labeling:

  • Antibody-based proximity labeling to identify TRIM4 interactors in their native cellular context

  • APEX2 or BioID fusion approaches to map the TRIM4 interactome

These methodological advances would enable more precise and comprehensive studies of TRIM4 biology, potentially revealing new functions and therapeutic opportunities that cannot be detected with current approaches .