TRIM65 Antibody, HRP conjugated

What is TRIM65 and what are its major structural domains?

TRIM65 is an E3 ubiquitin ligase belonging to the TRIM protein family. Its structure includes a conserved TRIM domain with a RING finger (responsible for E3 ligase activity), a B box-type zinc finger, a coiled-coil region, and a C-terminal SPRY domain. These domains enable TRIM65 to interact with various substrate proteins and mediate their ubiquitination . TRIM65 is encoded by a gene located on human chromosome 17q25.1 and is involved in multiple cellular processes including innate immunity, cancer progression, and inflammation regulation .

What applications are TRIM65 antibodies commonly used for?

TRIM65 antibodies are commonly used for:

• Western blotting (WB)

• Immunofluorescence (IF)

• Fluorescence-activated cell sorting (FACS)

• Immunohistochemistry (IHC)

• Enzyme-linked immunosorbent assay (ELISA)

The HRP-conjugated versions are particularly valuable for Western blot and ELISA applications where direct detection without secondary antibodies is beneficial for reducing background and improving specificity .

How do I properly validate a TRIM65 antibody before using it in my experiments?

Validation of a TRIM65 antibody should include:

• Western blot analysis using positive controls (tissues/cells known to express TRIM65) and negative controls (TRIM65 knockout samples or tissues with minimal expression)

• Testing reactivity across species if planning cross-species applications

• Comparing results with published literature

• Performing knockdown/knockout experiments to confirm specificity

• Testing different dilutions to optimize signal-to-noise ratio

For HRP-conjugated antibodies specifically, also validate that the conjugation doesn't affect binding specificity by comparing results with unconjugated versions .

What is the optimal protocol for using TRIM65 antibody in co-immunoprecipitation experiments?

For co-immunoprecipitation with TRIM65 antibody:

• Lysate preparation:

  • Harvest cells in non-denaturing lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA)

  • Include protease inhibitors and deubiquitinase inhibitors (like N-ethylmaleimide) if studying ubiquitination

  • Clear lysate by centrifugation (14,000 × g, 15 min, 4°C)

• Immunoprecipitation:

  • Pre-clear lysate with protein A/G beads for 1 hour

  • Incubate 500-1000 μg of lysate with 2-5 μg TRIM65 antibody overnight at 4°C

  • Add protein A/G beads for 2-4 hours

  • Wash 4-5 times with lysis buffer

  • Elute with 2× SDS sample buffer

For interacting partners like ARHGAP35 or TOX4, proximity ligation assays (PLA) may be more effective than standard IP, as demonstrated in colorectal cancer studies .

What are the key differences in protocol when using HRP-conjugated versus unconjugated TRIM65 antibodies?

The main protocol differences include:

For HRP-conjugated TRIM65 antibody:

• Direct detection without secondary antibody

• Typically requires lower primary antibody concentrations (1:1000-1:5000)

• Shorter protocol time as secondary antibody incubation is eliminated

• More sensitive to storage conditions (avoid freeze-thaw cycles)

• May have higher background in some applications, requiring optimization of blocking conditions

• Detection using chemiluminescent substrates directly after washing

For unconjugated TRIM65 antibody:

• Requires species-appropriate secondary antibody

• Usually used at 1:500-1:1000 dilutions

• Longer protocol with additional incubation and washing steps

• More flexible for dual-labeling experiments

• Often provides cleaner results in IHC applications

What are the best methods for studying TRIM65-mediated ubiquitination of target proteins?

Based on published research methodologies, the optimal approach includes:

• Cell-based ubiquitination assay:

  • Co-transfect cells with:

    • HA/His-tagged ubiquitin

    • FLAG-tagged TRIM65 (wild-type or RING domain mutant)

    • GFP/Myc-tagged substrate protein (e.g., MDA5, VCAM-1, ARHGAP35)

  • Treat with proteasome inhibitor (MG132, 10 μM) for 4-6 hours before harvest

  • Lyse cells under denaturing conditions (1% SDS buffer with heating)

  • Dilute lysate and immunoprecipitate substrate protein

  • Detect ubiquitination by Western blotting with anti-ubiquitin antibody

• Linkage-specific ubiquitination analysis:

  • Use ubiquitin mutants (K48-only, K63-only, K48R, K63R) to determine ubiquitin chain type

  • Alternatively, use linkage-specific antibodies (anti-K48-Ub, anti-K63-Ub)

  • This distinction is critical as K48-linked chains typically signal for degradation (as seen with TRIM65-VCAM-1 interaction) while K63-linked chains often regulate signaling (as with TRIM65-MDA5)

• In vitro ubiquitination assay:

  • Purify recombinant TRIM65, E1, E2 enzymes, and substrate

  • Combine with ATP, ubiquitin in reaction buffer

  • Incubate at 30°C for 1-2 hours

  • Analyze by SDS-PAGE and immunoblotting

For studying TRIM65's effect on substrate degradation rates, cycloheximide chase assays have proven effective, as demonstrated with ARHGAP35 and p53 .

How does TRIM65 function in the antiviral innate immune response?

TRIM65 plays a critical role in antiviral immunity, particularly in the MDA5-mediated pathway:

• MDA5 activation mechanism:

  • TRIM65 specifically interacts with the helicase domain of MDA5 (but not RIG-I)

  • It catalyzes K63-linked ubiquitination of MDA5 at lysine 743

  • This ubiquitination is essential for MDA5 oligomerization and activation

  • Activated MDA5 then signals through MAVS to induce interferon production

• Physiological evidence:

  • TRIM65-deficient mice show:

    • Complete blockage of EMCV-induced type I interferon production

    • Normal responses to VSV (which activates RIG-I pathway)

    • Increased susceptibility to EMCV infection

    • Inability to produce type I interferon in vivo during EMCV challenge

• Pathway specificity:

  • TRIM65 knockout specifically impairs HMW poly I:C and EMCV-RNA-induced interferon production

  • It has no effect on LMW poly I:C, 3pRNA, LPS, or adenovirus-induced responses

  • This confirms TRIM65's specific role in MDA5-dependent antiviral immunity

The specificity of TRIM65 for MDA5 (and not RIG-I) makes it a potential target for selective modulation of specific antiviral pathways.

What is the role of TRIM65 in cancer progression and what are the key substrates involved?

TRIM65's role in cancer is context-dependent, with evidence supporting oncogenic functions in multiple cancer types:

In Colorectal Cancer (CRC):

• TRIM65 is upregulated in CRC tissues compared to adjacent normal tissues

• High expression correlates with poor survival, metastasis, and recurrence

• Key substrate: ARHGAP35 (p190RhoGAP)

  • TRIM65 mediates K48-linked ubiquitination and degradation of ARHGAP35

  • This leads to elevated Rho GTPase activity

  • Results in increased formation of migration-related structures and enhanced metastasis

• TRIM65 overexpression enhances CRC cell proliferation, invasion, and migration

In Cervical Cancer:

• TRIM65 expression is significantly higher in cervical cancer tissues

• Promotes cell growth and migration

• Key substrate: p53

  • TRIM65 binds to p53 and promotes its ubiquitination and degradation

  • Accelerates p53 turnover as demonstrated by cycloheximide chase assays

  • This degradation can be blocked by proteasome inhibitor MG132

• TRIM65 also inhibits autophagy in cervical cancer cells

In Lung Cancer:

• Elevated in cisplatin-resistant non-small-cell lung cancer (NSCLC)

• Promotes autophagy and chemoresistance

• Mechanism: Induces ubiquitination and degradation of TNRC6A, resulting in suppressed expression of miR-138-5p

• Knockdown of TRIM65 enhances cisplatin-induced apoptosis and inhibits autophagy

How does TRIM65 regulate inflammatory responses?

TRIM65 functions as a critical regulator of inflammation through multiple mechanisms:

• VCAM-1 degradation pathway:

  • TRIM65 targets vascular cell adhesion molecule 1 (VCAM-1) for ubiquitination

  • It promotes K48-linked polyubiquitination of VCAM-1, leading to its degradation

  • This mechanism limits monocyte adherence to endothelium and tissue infiltration

  • TRIM65-deficient mice show:

    • Increased sensitivity to LPS-induced death

    • Sustained and severe pulmonary inflammation

    • Enhanced monocyte/macrophage infiltration into tissues

  • The protein levels of TRIM65 and VCAM-1 are inversely correlated during TNFα-induced endothelial activation

• Apoptosis regulation:

  • TRIM65 inhibits intestinal ischemia/reperfusion (II/R)-induced apoptosis

  • Mechanism: TRIM65 mediates K48-linked ubiquitination and degradation of TOX4

  • TRIM65-deficient mice show exacerbated II/R-induced intestinal apoptosis and damage

• Macrophage regulation:

  • LPS treatment decreases TRIM65 expression in macrophages and in C57BL/6J mice

  • This occurs through activation of the ERK1/2 signaling pathway

  • TRIM65 knockout promotes LPS-induced expression of inflammatory cytokines in macrophages

These findings suggest TRIM65 generally acts as a negative regulator of inflammation, making it a potential therapeutic target for inflammatory diseases.

How can I optimize TRIM65 detection in challenging samples with low expression?

For optimal detection of low TRIM65 expression:

• Signal amplification methods:

  • Use tyramide signal amplification (TSA) with HRP-conjugated antibodies

  • Apply antigen retrieval (citrate buffer pH 6.0, 95°C for 20 min) for fixed tissues

  • Implement sandwich ELISA with capture and detection antibodies for enhanced sensitivity

• Sample preparation optimization:

  • Enrich for TRIM65-expressing cellular compartments (primarily cytoplasmic, but also nuclear in some contexts)

  • Concentrate proteins using TCA precipitation or acetone precipitation before Western blot

  • For tissues, use laser capture microdissection to isolate specific cell populations

• Detection system enhancements:

  • Use high-sensitivity ECL substrates (SuperSignal West Femto)

  • Extend exposure times with incremental monitoring

  • Consider cooled CCD camera systems for digital Western blot imaging

• Antibody selection considerations:

  • Choose antibodies validated for the specific application and species

  • TRIM65 antibodies targeting the C-terminus (amino acids 419-508) have shown good specificity in human samples

  • Consider multiple antibodies targeting different epitopes for confirmation

What are the best approaches for studying changes in TRIM65 phosphorylation status?

Research has shown that TRIM65 undergoes phosphorylation that may regulate its activity. To study these modifications:

• Phosphorylation detection methods:

  • Phos-tag SDS-PAGE for mobility shift detection

  • Phospho-specific antibodies (when available)

  • Mass spectrometry for global phosphorylation site mapping

  • In vivo 32P-orthophosphate labeling for dynamic phosphorylation studies

• Key observations from literature:

  • Two discrete bands (57 kDa and 68 kDa) are observed when immunoblotting for TRIM65

  • The 68 kDa band is sensitive to calf intestinal alkaline phosphatase (CIAP) treatment, confirming it represents phosphorylated TRIM65

  • This phosphorylated form appears to decrease in tumor samples compared to matched normal tissues

  • Phosphorylation sites have been identified in colorectal cancer studies

• Experimental approach:

  • Treat cell lysates with phosphatase inhibitors to preserve phosphorylation

  • Split samples for parallel treatment with and without CIAP

  • Compare migration patterns on Western blot

  • For site identification, perform immunoprecipitation followed by mass spectrometry

• Functional analysis:

  • Generate phospho-mimetic and phospho-dead mutants at identified sites

  • Assess E3 ligase activity, substrate binding, and protein stability

  • Determine effects on cellular localization using immunofluorescence

What are the most effective experimental designs for comparing different TRIM65 splice variants or isoforms?

To effectively study TRIM65 splice variants or isoforms:

• Identification methods:

  • RT-PCR with primers spanning potential splice junctions

  • RNA-Seq analysis for comprehensive isoform discovery

  • 5' and 3' RACE to identify novel transcription start sites or polyadenylation sites

• Expression analysis:

  • Isoform-specific qPCR with primers/probes designed to unique exon junctions

  • Western blotting with antibodies targeting different domains

  • Create a comparative table of TRIM65 isoform expression across tissues:

  Tissue/Cell Type	Full-length TRIM65	Variant 1	Variant 2	Detection Method
  Endothelial cells	High	Minimal	Minimal	Western blot/qPCR
  Immune cells	Moderate	Low	Moderate	Western blot/qPCR
  Colorectal tissue	Variable (↑ in CRC)	Low	Low	IHC/Western blot
  Cervical tissue	Variable (↑ in CC)	Unknown	Unknown	IHC

• Functional characterization:

  • Clone individual isoforms into expression vectors

  • Create domain-specific deletion constructs

  • Compare:

    • Subcellular localization using fluorescent tags

    • Substrate binding affinity via co-IP or PLA

    • E3 ligase activity through in vitro ubiquitination assays

    • Effects on signaling pathways relevant to TRIM65 function

• CRISPR-based approaches:

  • Design isoform-specific knockouts by targeting unique exons

  • Use homology-directed repair to tag endogenous isoforms

  • Implement exon-specific knock-in of mutations to affect single isoforms

What are the common pitfalls when interpreting TRIM65 antibody results and how can they be avoided?

Common pitfalls and their solutions include:

• Non-specific bands in Western blot:

  • Cause: Cross-reactivity with other TRIM family members due to conserved domains

  • Solution: Include positive controls (overexpression) and negative controls (knockout/knockdown)

  • Always compare band pattern with expected molecular weight (57 kDa for unmodified TRIM65, 68 kDa for phosphorylated form)

• Variable results across different tissues/cells:

  • Cause: TRIM65 expression is tissue-dependent and regulated by stimuli like LPS and TNFα

  • Solution: Standardize experimental conditions and include appropriate context-specific controls

  • Monitor time-dependent changes, as TRIM65 levels fluctuate during activation of certain pathways

• Conflicting results between protein and mRNA levels:

  • Cause: Post-translational regulation, as TRIM65 itself undergoes ubiquitination

  • Solution: Always assess both protein and mRNA levels when possible

  • Use proteasome inhibitors to determine if discrepancies are due to protein degradation

• Antibody performance inconsistencies:

  • Cause: Batch-to-batch variation, different epitopes, conjugation effects

  • Solution: Validate each new antibody lot with known controls

  • For HRP-conjugated antibodies specifically, verify enzyme activity with substrate-only controls

• Difficulties detecting interaction partners:

  • Cause: Transient interactions or rapid degradation of substrates

  • Solution: Use proximity ligation assays instead of standard co-IP for detecting fleeting interactions (as shown with ARHGAP35)

  • Include proteasome inhibitors when studying substrate interactions

How do I reconcile contradictory data regarding TRIM65 function in different disease models?

To reconcile contradictory findings about TRIM65:

• Context-dependent functions:

  • TRIM65 exhibits tissue-specific and pathway-specific roles

  • In antiviral immunity: Positively regulates MDA5 pathway via K63-linked ubiquitination

  • In inflammation: Negatively regulates by targeting VCAM-1 for degradation via K48-linked ubiquitination

  • In cancer: Generally oncogenic but through distinct mechanisms in different cancer types

• Methodological approach to contradictions:

  • Create a comprehensive experimental framework that examines:

    • Cell/tissue type (endothelial vs. epithelial vs. immune cells)

    • Disease context (cancer vs. inflammation vs. viral infection)

    • Ubiquitination type (K48 vs. K63-linked chains)

    • Substrate-specific effects

• Analyzing discrepancies systematically:

  Context	TRIM65 Effect	Ubiquitin Type	Substrate	Cellular Outcome	Reference
  Viral infection	Activating	K63-linked	MDA5	Enhanced antiviral response
  Inflammation	Inhibitory	K48-linked	VCAM-1	Reduced adhesion molecule expression
  Colorectal cancer	Promoting	K48-linked	ARHGAP35	Enhanced migration and metastasis
  Cervical cancer	Promoting	K48-linked	p53	Reduced apoptosis
  Ischemia/reperfusion	Protective	K48-linked	TOX4	Reduced apoptosis

• Integration strategies:

  • Focus on identifying common molecular mechanisms across systems

  • Consider transcriptional, post-transcriptional, and post-translational regulation

  • Evaluate subcellular localization differences that might explain functional diversity

  • Account for feedback loops and compensatory mechanisms in experimental design

What are the key considerations when designing experiments to study TRIM65 E3 ligase activity in different biological contexts?

When designing experiments to study TRIM65 E3 ligase activity:

• Domain-specific functional analysis:

  • The RING domain is essential for E3 ligase activity

  • Create RING domain mutants as negative controls for ubiquitination activity

  • The coiled-coil and SPRY domains mediate substrate interactions

  • Use domain-specific deletions to map interaction regions with new substrates

• Substrate identification considerations:

  • Use proteomic approaches like iTRAQ for unbiased substrate screening

  • Verify physical interactions via co-IP, PLA, or yeast two-hybrid systems

  • Known substrates include MDA5, VCAM-1, ARHGAP35, p53, TOX4, TNRC6A

  • For new substrates, confirm direct binding and ubiquitination

• Context-specific experimental design:

  • For immune contexts: Study effects of viral mimics (poly I:C) or viral infection

  • For inflammatory contexts: Use TNFα, LPS, or ischemia/reperfusion models

  • For cancer contexts: Compare paired tumor/normal tissues and metastatic/primary samples

  • For each context, track both TRIM65 expression and activity

• Technical considerations for measuring E3 ligase activity:

  • Include appropriate E2 conjugating enzymes in in vitro assays

  • Use ubiquitin mutants (K48-only, K63-only) to determine chain specificity

  • Monitor substrate half-life via cycloheximide chase experiments

  • Employ proteasome inhibitors to distinguish between signaling vs. degradative ubiquitination

  • Consider global approaches like di-Gly remnant profiling for identifying ubiquitination sites

• In vivo validation approaches:

  • Use genetic models (TRIM65 knockout mice) for physiological relevance

  • Apply disease-relevant challenges (viral infection, inflammatory stimuli)

  • Measure functional outcomes beyond molecular changes

  • Consider conditional/inducible systems to avoid developmental effects