TRIM65 Antibody

What is TRIM65 and why is it significant in innate immunity studies?

TRIM65 is an E3 ubiquitin ligase belonging to the tripartite motif (TRIM) protein family. It plays a critical role in antiviral innate immunity by specifically interacting with melanoma differentiation-associated protein 5 (MDA5) and catalyzing its K63-linked ubiquitination. This post-translational modification is essential for MDA5 oligomerization and subsequent activation of type I interferon signaling pathways. TRIM65 functions as a critical component specifically in the MDA5 signaling pathway but does not affect RIG-I, TLR3, or cyclic GMP-AMP synthase signaling pathways. Studies using TRIM65-deficient mice have demonstrated increased susceptibility to encephalomyocarditis virus (EMCV) infection and inability to produce type I interferons in vivo, highlighting TRIM65's physiological importance in antiviral defense mechanisms .

What cellular processes can be effectively studied using TRIM65 antibodies?

TRIM65 antibodies enable researchers to investigate several critical cellular processes including:

• Protein-protein interactions between TRIM65 and MDA5, particularly through co-immunoprecipitation assays that reveal specific binding domains

• K63-linked ubiquitination events, especially those targeting MDA5 at lysine 743

• Protein oligomerization processes, particularly MDA5 aggregation following viral RNA sensing

• Activation of innate immune signaling cascades, including IRF3 phosphorylation and type I interferon production

• Subcellular localization patterns of TRIM65 and its binding partners during viral infection

TRIM65 antibodies allow visualization of these processes through various experimental approaches including Western blotting, immunofluorescence microscopy, immunoprecipitation, and chromatin immunoprecipitation depending on the specific research question .

How do I select the appropriate TRIM65 antibody for my experimental system?

When selecting a TRIM65 antibody for your research, consider these critical factors:

• Species reactivity: Verify that the antibody recognizes TRIM65 from your model organism. For example, some antibodies specifically detect human TRIM65 but may not cross-react with mouse or rat homologs.

• Application compatibility: Ensure the antibody is validated for your intended application (Western blot, immunoprecipitation, immunofluorescence, etc.). For instance, the H00201292-B01P antibody is specifically validated for Western blot applications with human samples.

• Antibody format: Consider whether you need a monoclonal or polyclonal antibody based on your experimental needs. Polyclonal antibodies like the mouse IgG format may provide higher sensitivity but potentially lower specificity.

• Epitope location: For studies focusing on specific TRIM65 domains (RING, B-box, coiled-coil, or SPRY), select antibodies targeting relevant regions. This is particularly important for studies examining domain-specific interactions, such as the SPRY domain's interaction with MDA5's helicase domain.

• Validation data: Review available experimental validation, including positive controls in cells known to express TRIM65 and negative controls in knockout systems .

How can TRIM65 antibodies be optimized for studying MDA5 ubiquitination processes?

To effectively study TRIM65-mediated ubiquitination of MDA5, researchers should implement the following optimization strategies:

• Combined immunoprecipitation approach: First immunoprecipitate MDA5 using anti-MDA5 antibodies, then probe for ubiquitination using anti-ubiquitin antibodies (preferably K63-linkage specific). This approach was successfully used to demonstrate that TRIM65 specifically catalyzes K63-linked ubiquitination of MDA5 at lysine 743.

• Ubiquitin mutant controls: Include experiments with ubiquitin mutants containing only K63 (K63O) or lacking K63 (K63R) to confirm K63-linkage specificity. Research has shown that TRIM65 efficiently catalyzes the linkage of K63O but not K63R ubiquitin mutants to MDA5.

• Domain mapping: For precise mechanistic studies, construct MDA5 domain mutants (e.g., 2CARD, helicase, and CTD domains) to identify which regions undergo TRIM65-mediated ubiquitination. Previous research identified that TRIM65 specifically ubiquitinates the helicase domain, particularly at lysine 743.

• In vitro reconstitution: Implement in vitro ubiquitination assays using purified components (UBE1, Ubc13/Uev1A, HA-tagged ubiquitin, DTT, and MgATP buffer) with immunopurified Flag-TRIM65 and Flag-MDA5 to directly assess enzymatic activity.

• Viral RNA stimulation: Include appropriate viral RNA stimulation (such as EMCV-RNA) to trigger physiologically relevant ubiquitination events that can be monitored with the antibodies .

What methodologies are recommended for detecting TRIM65-MDA5 interactions in different experimental systems?

For robust detection of TRIM65-MDA5 interactions across experimental systems, implement these methodological approaches:

• Co-immunoprecipitation (Co-IP): This technique effectively demonstrated the specific interaction between TRIM65 and MDA5 but not RIG-I. For optimal results, use mild lysis conditions (0.5% Triton X-100) to preserve protein-protein interactions, and include both forward (IP: TRIM65, WB: MDA5) and reverse (IP: MDA5, WB: TRIM65) approaches to confirm bidirectional interaction.

• Domain mapping through truncation mutants: Generate domain-specific constructs of both TRIM65 (RING, B-Box, coiled-coil, SPRY) and MDA5 (2CARD, helicase-N, helicase-C, CTD) to identify critical interaction interfaces. Research has established that the SPRY domain of TRIM65 specifically interacts with the helicase domain of MDA5.

• Confocal microscopy with fluorescent tags: Visualize protein colocalization using differentially tagged proteins (e.g., GFP-MDA5 and RFP-TRIM65). This approach revealed that TRIM65 colocalization promotes MDA5 aggregation, suggesting a role in oligomerization.

• Proximity ligation assay (PLA): For detecting endogenous protein interactions with high sensitivity, implement PLA using specific antibodies against TRIM65 and MDA5, which produces fluorescent signals only when proteins are in close proximity (<40 nm).

• Stimulation with appropriate ligands: Include treatments with high-molecular-weight poly I:C or EMCV-RNA, which enhance endogenous TRIM65-MDA5 interactions, as demonstrated in both HEK-293 and THP-1 cell models .

What techniques should be employed to study TRIM65-mediated MDA5 oligomerization?

TRIM65-mediated MDA5 oligomerization requires specialized techniques for accurate assessment:

• Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE): This technique is particularly effective for visualizing high-molecular-weight protein complexes and oligomers. The protocol involves:

  • Cell lysis with 0.5% Triton X-100 buffer

  • Sample preparation in 1× buffer containing 0.5× TBE, 10% glycerol, 2% SDS

  • Electrophoresis on 1.5% agarose gel in running buffer (1× TBE, 0.1% SDS) at 80V, 4°C

  • Transfer to membrane and immunoblotting with appropriate antibodies

• Size exclusion chromatography: For biochemical characterization of oligomer size and distribution, employ size exclusion columns to separate protein complexes by molecular weight, followed by Western blot analysis of collected fractions.

• Native PAGE: Unlike denaturing SDS-PAGE, native conditions preserve protein complexes and allow visualization of different oligomeric states using TRIM65 antibodies.

• Microscopy approaches: Utilize confocal or super-resolution microscopy to visualize MDA5 filament formation and aggregation in response to TRIM65 activity. Previous studies have shown that TRIM65 promotes MDA5 to form visible aggregates in HeLa cells.

• Mutational analysis: Include critical controls such as the MDA5 K743R mutation, which blocks TRIM65-mediated ubiquitination and subsequent oligomerization, thus preventing downstream signaling activation .

What are the recommended protocols for TRIM65 immunoprecipitation to study protein interactions?

For optimal TRIM65 immunoprecipitation to study protein interactions, follow these methodological guidelines:

• Lysis buffer composition: Use a gentle lysis buffer containing 0.5% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 1 mM PMSF, and protease inhibitor cocktail. This preserves protein-protein interactions while effectively solubilizing membrane-associated proteins.

• Pre-clearing step: Incubate lysates with protein A/G beads for 1 hour at 4°C to reduce non-specific binding, followed by collection of the supernatant for immunoprecipitation.

• Antibody incubation conditions: For endogenous TRIM65 immunoprecipitation, incubate cleared lysates with 2-5 μg anti-TRIM65 antibody overnight at 4°C with gentle rotation. For tagged TRIM65 (e.g., Flag-TRIM65), use the appropriate anti-tag antibody following the same conditions.

• Bead selection and handling: Add protein A/G magnetic or agarose beads to antibody-lysate mixtures and incubate for 2-4 hours at 4°C. Wash beads 4-5 times with lysis buffer to remove non-specific interactions.

• Elution strategies: For subsequent Western blot analysis, elute immune complexes by boiling in SDS sample buffer. For applications requiring native protein (e.g., in vitro ubiquitination assays), elute with Flag peptide (if using Flag-tagged TRIM65) or mild acid elution for endogenous proteins.

• RNA-enhanced interactions: To capture physiologically relevant interactions, consider stimulating cells with high-molecular-weight poly I:C or EMCV-RNA prior to lysis, which enhances TRIM65-MDA5 interactions .

How should Western blot conditions be optimized for TRIM65 detection?

For optimal TRIM65 detection by Western blot, implement these technical considerations:

• Sample preparation:

  • Include protein phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄) and deubiquitinase inhibitors (N-ethylmaleimide) in lysis buffers

  • Use RIPA buffer for total protein extraction or Triton X-100 buffer for preserving protein complexes

  • Sonicate samples briefly to shear DNA and reduce sample viscosity

• Gel selection and transfer parameters:

  • Use 8-10% SDS-PAGE gels for optimal resolution of TRIM65 (approximately 65 kDa)

  • For ubiquitinated forms, use 6-8% gels to better resolve high-molecular-weight species

  • Transfer to PVDF membranes (rather than nitrocellulose) at 100V for 90 minutes or 30V overnight at 4°C for high-molecular-weight proteins

• Blocking and antibody incubation:

  • Block membranes with 5% non-fat dry milk in TBST for standard applications

  • For phospho-specific detection, use 5% BSA in TBST instead of milk

  • Incubate with primary antibody (1:500-1:2000 dilution) overnight at 4°C

  • Use conformation-specific secondary antibodies to avoid detecting denatured IgG from immunoprecipitations

• Signal enhancement strategies:

  • Implement enhanced chemiluminescence (ECL) detection systems

  • For weak signals, consider using signal enhancers or HRP-conjugated polymer detection systems

  • For quantitative analysis, consider fluorescent secondary antibodies and imaging systems

• Controls and validation:

  • Include positive controls (cells known to express TRIM65) and negative controls (TRIM65-knockout samples)

  • For verification of specificity, pre-incubate antibody with blocking peptide .

What experimental approaches are recommended for investigating TRIM65's role in antiviral responses?

To comprehensively investigate TRIM65's role in antiviral responses, implement these experimental approaches:

• Knockout and reconstitution systems:

  • Generate TRIM65-deficient cells using CRISPR/Cas9 or obtain TRIM65-knockout mouse models

  • Perform rescue experiments by reconstituting knockout systems with wild-type TRIM65 or domain mutants (particularly ΔSPRY domain)

  • Compare antiviral responses between wild-type, knockout, and reconstituted systems following viral challenge

• Viral infection models:

  • Use EMCV as the primary viral model, as it specifically activates the MDA5-TRIM65 pathway

  • Include VSV as a control virus that activates RIG-I-dependent (TRIM65-independent) pathways

  • Monitor viral replication using plaque assays or qPCR for viral genomes

  • Examine tissue damage in animal models through histopathological analysis

• Cytokine production assessment:

  • Measure type I interferon (IFN-α, IFN-β) production using ELISA or bioassays

  • Quantify interferon-stimulated gene expression (ISG15, ISG56) through qRT-PCR

  • Analyze IRF3 phosphorylation status by Western blot as a direct readout of pathway activation

• Ligand-specific stimulation:

  • Use high-molecular-weight poly I:C as a specific MDA5 agonist

  • Include appropriate controls: low-molecular-weight poly I:C (RIG-I agonist), 5'-triphosphate RNA (RIG-I agonist), extracellular LPS (TLR4 agonist)

  • Compare responses to viral RNA isolated from EMCV-infected or VSV-infected cells

• In vivo validation:

  • Challenge TRIM65-knockout mice with EMCV

  • Monitor survival rates, viral titers in target organs, and serum interferon levels

  • Perform histopathological analysis of infected tissues .

How can researchers address weak or inconsistent TRIM65 antibody signals in Western blots?

When encountering weak or inconsistent TRIM65 signals in Western blots, implement these troubleshooting strategies:

• Sample preparation optimization:

  • Increase protein concentration (50-100 μg total protein per lane)

  • Add protease inhibitors freshly before lysis

  • Avoid repeated freeze-thaw cycles of protein samples

  • Include phosphatase inhibitors to preserve post-translational modifications

• Antibody-specific adjustments:

  • Optimize primary antibody concentration through titration experiments (1:250 to 1:2000)

  • Extend primary antibody incubation time (overnight at 4°C instead of 1-2 hours)

  • Test different antibody clones or lots if available

  • Consider using a more sensitive detection system (HRP-polymer-based or fluorescent-based)

• Technical modifications:

  • Increase transfer time or reduce voltage for more efficient protein transfer

  • Use freshly prepared buffers and reagents

  • For challenging samples, consider specialized membrane activation procedures

  • Test different blocking agents (BSA vs. milk) to reduce background while preserving signal

• Protein enrichment approaches:

  • For low abundance targets, concentrate proteins through immunoprecipitation before Western blot

  • Use subcellular fractionation to enrich for TRIM65-containing compartments

  • Consider using protein concentration methods (TCA precipitation, acetone precipitation)

• Expression-enhancing treatments:

  • Stimulate cells with appropriate ligands (poly I:C, viral RNA) to upregulate TRIM65 expression

  • For pathway studies, synchronize cells in specific cell cycle phases to normalize expression levels .

What strategies help optimize detection of TRIM65-mediated ubiquitination events?

For optimal detection of TRIM65-mediated ubiquitination events, implement these specialized approaches:

• Deubiquitinase inhibition:

  • Add N-ethylmaleimide (5-10 mM) to lysis buffers to inhibit deubiquitinases

  • Include ubiquitin aldehyde (1-5 μM) in lysates to preserve ubiquitin chains

  • Perform rapid sample processing at 4°C to minimize enzymatic degradation

• Denaturing conditions for ubiquitination analysis:

  • Use hot SDS lysis buffer (1% SDS, 5 mM EDTA, 10 mM DTT, heated to 95°C) to disrupt non-covalent interactions

  • Dilute SDS lysates 10-fold with non-denaturing buffer before immunoprecipitation

  • Perform tandem ubiquitin binding entity (TUBE) pulldowns to enrich for ubiquitinated proteins

• Ubiquitin linkage-specific approaches:

  • Employ K63-linkage specific antibodies for immunoblotting

  • Use ubiquitin mutant constructs (K63O, K63R) as controls to confirm linkage specificity

  • Include wild-type and lysine-to-arginine mutants of target proteins (e.g., MDA5 K743R)

• In vitro systems for mechanism validation:

  • Reconstitute ubiquitination reactions with purified components (E1, E2, E3 ligases)

  • Implement cell-free assays with immunopurified components to directly assess enzymatic activity

  • Use recombinant proteins with defined mutations to confirm specific ubiquitination sites

• Controls and verification:

  • Compare ubiquitination patterns between wild-type and TRIM65-deficient cells

  • Include proteasome inhibitors (MG132) when studying K48-linked ubiquitination

  • Verify results using mass spectrometry to identify precise ubiquitination sites and linkage types .

How can researchers differentiate between TRIM65 and other TRIM family proteins in experimental systems?

To effectively differentiate TRIM65 from other TRIM family proteins and avoid cross-reactivity issues, implement these methodological approaches:

• Antibody selection and validation:

  • Choose antibodies raised against unique regions of TRIM65 (particularly C-terminal regions) rather than conserved domains

  • Perform validation using TRIM65-knockout systems as negative controls

  • Test for cross-reactivity against recombinant TRIM family proteins, especially closely related members

  • Consider using epitope-tagged TRIM65 constructs for highly specific detection

• Expression analysis controls:

  • Include siRNA/shRNA knockdown controls targeting TRIM65-specific sequences

  • Implement qRT-PCR with primers targeting unique TRIM65 regions to confirm specificity at the mRNA level

  • Compare expression patterns across multiple cell types with known TRIM65 expression profiles

• Functional differentiation approaches:

  • Leverage the specific interaction between TRIM65 and MDA5 (not shared by other TRIM proteins)

  • Utilize EMCV infection models, which specifically depend on TRIM65 but not other TRIM proteins

  • Employ domain-specific constructs that highlight unique functions of TRIM65

• Advanced techniques for specificity:

  • Use custom antibodies raised against synthetic peptides unique to TRIM65

  • Implement immunodepletion experiments with one antibody followed by detection with another

  • Consider mass spectrometry-based approaches for unambiguous protein identification

• Bioinformatic prediction tools:

  • Utilize sequence alignment tools to identify unique epitopes for antibody generation

  • Employ structural prediction models to identify accessible regions specific to TRIM65

  • Design experiments based on predicted post-translational modifications unique to TRIM65 .

What are the best experimental controls for validating TRIM65 antibody specificity?

To rigorously validate TRIM65 antibody specificity and ensure reliable experimental results, implement these essential controls:

• Genetic knockout/knockdown validation:

  • Test antibodies in TRIM65-knockout cells or tissues (CRISPR/Cas9-generated or from knockout mice)

  • Include siRNA or shRNA-mediated TRIM65 knockdown samples to demonstrate signal reduction

  • Complement with rescue experiments showing signal restoration upon TRIM65 re-expression

• Overexpression controls:

  • Compare endogenous TRIM65 detection with overexpressed wild-type TRIM65

  • Include tagged TRIM65 constructs that can be detected with both anti-TRIM65 and anti-tag antibodies

  • Test truncated TRIM65 constructs of defined molecular weights to confirm correct band identification

• Peptide competition assays:

  • Pre-incubate antibody with the immunizing peptide before application to block specific binding

  • Include gradient concentrations of blocking peptide to demonstrate dose-dependent signal reduction

  • Use unrelated peptides as negative controls to confirm specificity of competition

• Cross-species reactivity assessment:

  • Test antibodies against TRIM65 from multiple species when working with diverse experimental models

  • Compare detection patterns in cells from different species with known TRIM65 sequence homology

  • Include species-specific positive controls when validating antibodies for cross-reactivity

• Multiple antibody validation:

  • Compare detection patterns using antibodies targeting different TRIM65 epitopes

  • Perform sequential immunoprecipitation with different antibodies to confirm target identity

  • Conduct parallel assays using monoclonal and polyclonal antibodies for comprehensive validation .

How can TRIM65 antibodies be employed to investigate its potential roles beyond antiviral immunity?

While TRIM65 is primarily studied in antiviral immunity contexts, researchers can expand investigations into other potential functions using these approaches:

• Protein interaction network mapping:

  • Perform immunoprecipitation with TRIM65 antibodies followed by mass spectrometry to identify novel binding partners

  • Conduct yeast two-hybrid screens using TRIM65 domains as bait to discover context-specific interactions

  • Implement proximity labeling approaches (BioID, APEX) coupled with TRIM65 antibody validation to map spatial protein networks

• Cell-type specific expression profiling:

  • Employ immunohistochemistry with TRIM65 antibodies across diverse tissue panels

  • Combine with lineage-specific markers to identify cell populations with high TRIM65 expression

  • Analyze expression in disease states compared to healthy tissues to identify dysregulation patterns

• Post-translational modification landscape:

  • Use TRIM65 antibodies for immunoprecipitation followed by mass spectrometry to identify modifications

  • Develop modification-specific antibodies (phospho-TRIM65, acetyl-TRIM65) for signaling studies

  • Investigate crosstalk between TRIM65's own modifications and its ubiquitin ligase activity

• Subcellular localization dynamics:

  • Implement super-resolution microscopy with TRIM65 antibodies to track subcellular distribution

  • Perform fractionation studies followed by Western blotting to quantify compartment-specific localization

  • Investigate stimulus-dependent translocation using live-cell imaging techniques

• Non-canonical functions exploration:

  • Investigate potential roles in cell cycle regulation, differentiation, or stress responses

  • Examine TRIM65 expression and activity in cancer models or neurodegenerative diseases

  • Explore possible functions in transcriptional regulation through chromatin immunoprecipitation approaches .

What emerging techniques might enhance TRIM65 antibody applications in research?

Emerging technologies offer exciting opportunities to enhance TRIM65 antibody applications:

• Single-cell protein analysis:

  • Adapt TRIM65 antibodies for mass cytometry (CyTOF) to quantify expression at single-cell resolution

  • Implement imaging mass cytometry for spatial distribution of TRIM65 in tissue contexts

  • Develop single-cell Western blot protocols for TRIM65 quantification in heterogeneous populations

• Advanced imaging approaches:

  • Apply expansion microscopy with TRIM65 antibodies for nanoscale resolution of protein complexes

  • Implement light-sheet microscopy for whole-tissue imaging of TRIM65 distribution

  • Utilize lattice light-sheet microscopy for dynamic tracking of TRIM65 in living cells

• Proximity-dependent methodologies:

  • Develop split-protein complementation assays to monitor TRIM65-MDA5 interactions in real-time

  • Adapt TRIM65 antibodies for proximity ligation assays to visualize specific protein interactions

  • Implement FRET-based biosensors to monitor TRIM65 conformational changes upon activation

• Cryo-electron microscopy applications:

  • Use TRIM65 antibodies for immunogold labeling in cryo-EM studies of protein complexes

  • Implement correlative light and electron microscopy to link functional data with structural insights

  • Develop nanobody-based probes derived from TRIM65 antibodies for improved sample penetration

• Multiplexed detection systems:

  • Adapt TRIM65 antibodies for multiplexed ion beam imaging (MIBI) for simultaneous profiling

  • Implement DNA-barcoded antibody technologies for highly multiplexed protein detection

  • Develop spatial transcriptomics approaches combined with TRIM65 protein detection .