TRIM3 Antibody, FITC conjugated

What is TRIM3 and why is it significant in scientific research?

TRIM3, also known as RING finger protein 22 (RNF22), RING finger protein 97 (RNF97), or brain-expressed RING finger protein (BERP), is a 744 amino acid protein belonging to the TRIM family characterized by the RING-B-box coiled-coil (RBCC) structure . TRIM3 functions as a tumor suppressor by regulating p21Waf1/Cip1 . Additionally, TRIM3 plays crucial roles in cell motility and cargo transport by interacting with key components of the actin cytoskeleton, such as α-actinin-4 and myosin V . Recent research also indicates TRIM3's involvement in attenuating cytokine storms during viral infections by promoting TLR3 degradation and inhibiting the MAPK signaling pathway . This multifunctional nature makes TRIM3 a significant target in cancer research, immunology, and cell biology.

What are the key structural domains of TRIM3 and how do they relate to function?

TRIM3 contains several functional domains that contribute to its diverse biological activities:

Research demonstrates that the NHL domain is necessary but not sufficient for p21 binding, while α-actinin-4 binds to the RBCC domain and myosin V interacts with the C-terminal β-propeller domain . These domain-specific interactions highlight the importance of targeting specific regions when using antibodies for different research applications.

How does TRIM3 function as a tumor suppressor?

TRIM3 functions as a tumor suppressor primarily through its interaction with p21Waf1/Cip1. Research indicates that TRIM3 can bind to p21 and sequester it away from cyclin D1-cdk4 complexes, thereby reducing cell proliferation . When TRIM3 expression is reduced, p21 accumulates and can promote tumor growth in certain contexts, particularly in proneural glioblastomas . Experiments show that TRIM3 and cyclin-cdk complexes compete for binding to p21, with TRIM3 binding preferentially to p21 that is not associated with cyclin-cdk complexes . The growth suppressive activity of TRIM3 is inseparable from its ability to bind p21, as demonstrated by mutational studies where NHL domain-deficient TRIM3 was unable to induce growth arrest .

What are the optimal methods for detecting TRIM3 expression in cell and tissue samples?

Based on the available research, several methods have proven effective for detecting TRIM3:

• Western Blotting: Using specific TRIM3 antibodies at a concentration of 200 μg/ml provides reliable detection of TRIM3 protein . When normalizing expression, consider using appropriate controls such as GAPDH or, in the case of oligodendrocyte-rich samples, olig2 expression .

• Immunoprecipitation: TRIM3 can be successfully immunoprecipitated using antibodies targeting amino acids 19-128 . This approach is particularly useful for studying TRIM3's interactions with binding partners like p21 .

• Immunofluorescence: TRIM3 is primarily localized to cytoplasmic filaments, making immunofluorescence an effective technique for visualizing its subcellular distribution . When using FITC-conjugated antibodies, consider that the excitation/emission maxima wavelengths are typically around 495 nm/524 nm .

• qRT-PCR: For mRNA expression analysis, qRT-PCR has been successfully used to detect TRIM3 mRNA levels in various tissues including liver, spleen, lung, and kidney, as well as in peripheral blood mononuclear cells (PBMCs) .

How should experiments be designed to study TRIM3's interaction with p21?

When investigating TRIM3's interaction with p21, consider the following experimental design principles:

• Co-immunoprecipitation: Endogenous TRIM3 and p21 can be co-immunoprecipitated from densely grown cell cultures . Both proteins can also be co-precipitated when full-length TRIM3 is overexpressed in appropriate cell lines .

• Domain-specific analysis: Include TRIM3 mutants lacking specific domains (RING, B-box, coiled-coil, ABP, or NHL) to determine which regions are necessary for p21 binding. Research has shown that the NHL domain is necessary but not sufficient for this interaction .

• Competition assays: Design experiments that account for competing interactions, as research shows that cyclin-cdk binding sequesters p21 from TRIM3 . Using mutants of p21 deficient in binding cyclin-cdk complexes can help elucidate these competitive interactions .

• Recombinant protein studies: The interaction between TRIM3 and p21 can be reconstituted using recombinant proteins produced in E. coli, providing a system to study direct binding without cellular confounding factors .

What are the best practices for using FITC-conjugated antibodies in flow cytometry applications targeting TRIM3?

While specific data for FITC-conjugated TRIM3 antibodies is limited in the search results, general best practices for flow cytometry with FITC-conjugated antibodies include:

• Sample preparation: For cell lines or primary cells, ensure proper fixation and permeabilization since TRIM3 is primarily a cytoplasmic protein .

• Controls: Include appropriate isotype controls matched to the host species and immunoglobulin class of the primary antibody. For TRIM3 antibodies derived from mouse IgG1 κ, use matching isotype controls .

• Signal optimization: FITC has excitation/emission maxima at approximately 495 nm/524 nm . Ensure your flow cytometer is properly calibrated for this fluorophore.

• Multiparameter analysis: When studying TRIM3 in immune cell populations, consider co-staining with lineage markers. This approach was successfully used in studies examining TLR3 expression on peripheral blood monocytes in relation to TRIM3 function .

• Data analysis: When analyzing flow cytometry data, gate on single cells first, then identify positive populations based on fluorescence intensity compared to isotype controls.

How can TRIM3 antibodies be utilized to investigate its role in immune response modulation?

Research indicates that TRIM3 attenuates cytokine storms during viral infections by promoting TLR3 degradation and inhibiting the MAPK signaling pathway . To investigate this role:

• Expression analysis: Use TRIM3 antibodies for western blotting or flow cytometry to compare expression levels between healthy controls and patients with inflammatory conditions. Research has shown differential TRIM3 mRNA expression in PBMCs from SFTS patients compared to healthy controls .

• Protein-protein interaction studies: Employ co-immunoprecipitation with TRIM3 antibodies to identify interactions with immune signaling components. Previous research has investigated interactions between TRIM3 and TLR3 using this approach .

• Signaling pathway analysis: Combine TRIM3 antibodies with antibodies against phosphorylated components of the MAPK pathway (p-p38, p-JNK1/2, p-IKKα/β, p-NF-κB) to examine how TRIM3 modulates signaling cascades during immune responses .

• Functional studies: Compare cytokine production in TRIM3 overexpression and knockout models, measuring levels by ELISA or qRT-PCR as was done in research with THP-1 cells and peritoneal macrophages infected with Dabie bandavirus .

What methodological approaches are effective for studying TRIM3's role in the ubiquitin-proteasome pathway?

Given TRIM3's RING domain and potential E3 ubiquitin ligase activity, several approaches can be effective:

• Ubiquitination assays: Use TRIM3 antibodies in conjunction with ubiquitin antibodies (particularly K48-linked ubiquitin, which was examined in previous research ) to detect ubiquitinated target proteins.

• Proteasomal inhibition experiments: Compare protein levels with and without proteasome inhibitors to determine if TRIM3's effects on target proteins (such as TLR3) are proteasome-dependent .

• Domain mutant studies: Create RING domain mutants of TRIM3 to determine if this domain is essential for the ubiquitination and degradation of target proteins.

• In vitro ubiquitination: Reconstitute the ubiquitination reaction using purified components including recombinant TRIM3, E1, E2, ubiquitin, and potential substrates to demonstrate direct enzymatic activity.

These approaches can help elucidate whether TRIM3 directly mediates ubiquitination and degradation of target proteins or functions through other mechanisms.

What are common issues when working with TRIM3 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with TRIM3 antibodies:

• Multiple bands on western blots: TRIM3 has three named isoforms resulting from alternative splicing . Additionally, post-translational modifications may cause size variations. Solution: Use positive controls with known TRIM3 expression and refer to expected molecular weights for different isoforms.

• Low signal intensity: TRIM3 may be expressed at low levels in some tissues or cell types. Solution: Optimize protein loading, increase antibody concentration or incubation time, or consider using more sensitive detection methods.

• Non-specific binding: This can obscure true TRIM3 signal. Solution: Optimize blocking conditions, titrate primary antibody concentration, and include appropriate negative controls such as TRIM3 knockout or knockdown samples .

• Immunoprecipitation inefficiency: Solution: Verify antibody binding capacity, optimize lysis buffer composition, and consider using agarose-conjugated TRIM3 antibodies for improved efficiency .

How should experimental conditions be optimized when studying TRIM3 in different cell types?

Different cell types may require specific optimization strategies:

• Immune cells: For THP-1 cells and PBMCs, research has shown successful TRIM3 detection following DBV infection at MOI = 0.5 for 2 hours, with cell viability of 98.9% . This provides a starting point for optimization in immune cell models.

• Neural cells: In glioblastoma models, TRIM3 expression and function have been studied using the YH/J12 and T98G cell lines . These models may require specific culture conditions to achieve appropriate TRIM3 expression levels.

• Primary cells: For primary cells such as peritoneal macrophages from TRIM3 knockout mice, special consideration should be given to extraction and culture conditions to maintain cellular viability and phenotype .

• Tissue samples: When examining TRIM3 expression in tissue samples, immunofluorescence has been used successfully. Proper fixation and antigen retrieval protocols should be optimized for each tissue type .

How can TRIM3 antibodies be integrated into high-throughput screening approaches?

TRIM3 antibodies can be incorporated into high-throughput screening through several methodologies:

• Protein microarrays: Immobilize TRIM3 antibodies on microarray platforms to screen for interacting proteins or to evaluate TRIM3 expression across multiple samples simultaneously.

• High-content imaging: Use FITC-conjugated TRIM3 antibodies in automated microscopy systems to quantify TRIM3 expression, subcellular localization, and co-localization with other proteins across many conditions.

• Flow cytometry-based screening: Employ FITC-conjugated TRIM3 antibodies in flow cytometry to screen cell populations for TRIM3 expression following various treatments or genetic modifications.

• CRISPR screens: Combine CRISPR screening with TRIM3 antibody detection to identify genes that regulate TRIM3 expression or function in various biological contexts.

These approaches facilitate systematic investigation of TRIM3 biology across multiple experimental conditions, potentially uncovering novel regulatory mechanisms and functions.

What are emerging applications for studying TRIM3 in neurodegenerative diseases?

Given TRIM3's expression in the brain (as indicated by its alternate name, brain-expressed RING finger protein or BERP ) and its interactions with the cytoskeleton, several emerging applications exist:

• Protein aggregation studies: Investigate whether TRIM3 plays a role in protein quality control by targeting misfolded proteins for degradation in neurodegenerative disease models.

• Axonal transport analysis: Examine TRIM3's interaction with myosin V in the context of axonal transport, which is often disrupted in neurodegenerative conditions.

• Neuroinflammation models: Given TRIM3's role in immune modulation , explore its function in neuroinflammatory processes associated with neurodegenerative diseases.

• Cytoskeletal dynamics: Investigate how TRIM3's interactions with the actin cytoskeleton might influence neuronal morphology and function in disease states.

These applications represent frontier areas where TRIM3 research might yield insights into pathological mechanisms and potential therapeutic targets.

How do different antibody formats for TRIM3 detection compare in research applications?

Based on available information, different TRIM3 antibody formats offer distinct advantages:

When selecting an antibody format, researchers should consider their specific application requirements, detection systems available, and experimental design constraints.

What methodological differences should be considered when comparing TRIM3 expression across different disease models?

When comparing TRIM3 expression across disease models, several methodological considerations are important:

• Standardization: Use consistent antibody concentrations, incubation times, and detection methods across all samples to ensure comparability.

• Appropriate controls: Include both positive and negative controls specific to each disease model. For viral infection models, uninfected samples serve as controls , while for tumor studies, matched normal tissue provides the appropriate comparison .

• Quantification methods: Implement rigorous quantification approaches, such as normalizing TRIM3 levels to appropriate housekeeping proteins or calculating relative expression using qRT-PCR with validated reference genes .

• Cell type considerations: Account for tissue heterogeneity, as TRIM3 expression may vary between cell types within a tissue. In the RCAS-PDGF-HA/nestin-TvA model, researchers normalized to olig2 expression to account for oligodendrocyte contribution .

• Time course analysis: Consider temporal dynamics, as TRIM3 expression and function may change during disease progression or treatment response.

These methodological considerations ensure robust and reproducible comparisons of TRIM3 expression across diverse experimental systems and disease models.