TRIM54 Antibody

What is TRIM54 and where is it primarily expressed?

TRIM54 (Tripartite motif-containing protein 54), also known as MURF, MURF3, or RNF30, is a protein primarily expressed in heart and skeletal muscle tissues. It plays crucial roles in skeletal myoblast differentiation and myotube fusion . Expression analysis shows that TRIM54 is specifically expressed in cardiac and skeletal muscle, with molecular weights of approximately 40-45 kDa depending on the isoform . Research has demonstrated that TRIM54 may bind and stabilize microtubules during myotube formation, suggesting its importance in muscle development .

What applications are TRIM54 antibodies validated for?

TRIM54 antibodies have been validated for multiple research applications including:

This data is compiled from multiple antibody validation studies .

How should TRIM54 antibodies be stored to maintain reactivity?

For optimal antibody performance, TRIM54 antibodies should be stored at -20°C for long-term preservation. They typically remain stable for one year after shipment when properly stored. The antibodies are usually supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, or in Tris Buffered Saline with 0.5% BSA and 0.02% Sodium Azide .

When working with TRIM54 antibodies, it's important to avoid repeated freeze/thaw cycles. For antibodies with high concentration (e.g., 500 μg/ml), aliquoting upon receipt is recommended, although some manufacturers note that aliquoting is unnecessary for -20°C storage for certain formulations .

How can I optimize TRIM54 antibody use for studying its role in tendinopathy models?

Recent research has revealed that TRIM54 plays a critical role in tendinopathy by alleviating inflammation and apoptosis through stabilization of YOD1 . When designing experiments to study this:

• In vitro model selection: Use tendon-derived stem cells (TDSCs) exposed to TNF-α to create an inflammation model. This approach has been validated to significantly decrease TRIM54 expression while increasing inflammatory markers and apoptosis .

• Experimental design:

  • Set up TRIM54 overexpression models using appropriate vectors

  • Confirm overexpression using qRT-PCR and Western blotting

  • Assess downstream effects on:

    • Tenogenesis markers (COL1, collagen type 3, scleraxis, Tnmd)

    • Progenitor markers (CD146, Sox2, Oct4)

    • Inflammatory cytokines (IL-1β, IL-6)

    • Apoptotic markers (Bax, c-caspase-3)

• Validation methods: Use cycloheximide chase assays and immunoprecipitation to study the interaction between TRIM54 and YOD1. Co-immunoprecipitation studies should include appropriate tagged proteins (such as Myc-tagged YOD1 and Flag-tagged TRIM54) .

What are the optimal antigen retrieval methods for TRIM54 immunohistochemistry?

For successful immunohistochemical detection of TRIM54:

• Primary recommendation: Use TE buffer pH 9.0 for antigen retrieval . This has been validated in human heart tissue and human skeletal muscle tissue.

• Alternative method: Citrate buffer pH 6.0 can also be effective for TRIM54 antigen retrieval .

• Tissue-specific considerations: For tendinopathy studies, normal tendon and tendinopathic tendon tissues require careful optimization. Studies have shown decreased TRIM54 staining in tendinopathic tendons compared to normal tendons, so staining protocols must be sensitive enough to detect these differences .

• Controls: Always include appropriate positive control tissues (heart and skeletal muscle) and negative controls (tissues known not to express TRIM54) to validate staining specificity.

How can I study TRIM54's role in ubiquitination pathways?

TRIM54 functions as an E3 ligase that can target proteins for ubiquitination and subsequent degradation. To study this function:

• Protein interaction analysis:

  • Perform co-immunoprecipitation (Co-IP) assays with tagged TRIM54 (e.g., Flag-tagged) and potential target proteins

  • Use HEK 293T cells for transfection and expression of tagged proteins

• Ubiquitination assessment:

  • Conduct cycloheximide chase assays to determine protein stability in the presence/absence of TRIM54

  • For identified interactions (e.g., TRIM54 and YOD1 or TRIM54 and Axin1), monitor protein levels over time

• Functional validation:

  • Implement TRIM54 overexpression or knockdown strategies

  • Assess downstream pathway activation (e.g., Wnt/β-catenin signaling in hepatocellular carcinoma models)

The TRIM54-Axin1-β-catenin axis has been shown to play a critical role in hepatocellular carcinoma progression, providing a model system for studying TRIM54's ubiquitination function .

What are the methodological considerations when analyzing discrepancies in TRIM54 molecular weight?

Researchers often observe variations in TRIM54 molecular weight across different experimental systems:

• Expected molecular weights:

  • Calculated molecular weight: 358 aa, 40 kDa

  • Observed molecular weights: 40-45 kDa

  • Some reports indicate detection of two isoforms (45 kDa and 38 kDa)

• Tissue-specific variations:

  • In mouse liver tissue, typically observed at 41 kDa

  • In HEK293, PC3, and U87 cell lysates and rat brain tissue, observed at both 41 kDa and 90 kDa

• Methodological approach to resolving discrepancies:

  • Use multiple antibodies targeting different epitopes

  • Perform TRIM54 knockdown or overexpression to confirm specificity

  • Consider tissue-specific post-translational modifications

  • Use appropriate positive controls from tissues known to express TRIM54 (heart and skeletal muscle)

How can I design experiments to evaluate TRIM54's role in cancer progression?

Based on studies showing TRIM54 overexpression in hepatocellular carcinoma (HCC) and its association with poor patient outcomes :

How can I validate TRIM54 antibody specificity for my experimental system?

To ensure antibody specificity when working with TRIM54:

• Positive control selection:

  • Use tissues with known high TRIM54 expression (heart and skeletal muscle)

  • Validated cell lines include HepG2 (for IF/ICC) and HEK293 (for WB)

• Specificity validation strategies:

  • Perform Western blot with TRIM54 knockdown or overexpression samples

  • Pre-incubate antibody with immunizing peptide (if available) to confirm specific binding

  • Compare staining patterns across multiple antibodies targeting different TRIM54 epitopes

• Downstream validation:

  • For functional studies, validate phenotypic changes with multiple TRIM54-targeting shRNAs

  • Rescue experiments by expressing shRNA-resistant TRIM54 constructs

What are the key considerations when designing Western blot experiments for TRIM54?

For optimal Western blot results with TRIM54 antibodies:

• Sample preparation:

  • For tissue samples: heart, skeletal muscle, and liver tissues have been validated

  • For cell lines: HEK293, PC3, U87, and HepG2 cells have shown detectable expression

• Loading controls:

  • α-tubulin is commonly used for cytoplasmic fraction

  • p84 has been validated for nuclear fraction analysis

• Technical parameters:

  • Working dilutions: 1:2000-1:16000 for most commercial antibodies

  • Expected bands: 40-45 kDa (primary) with possible additional bands at ~90 kDa in some samples

  • Secondary antibody selection: Anti-rabbit IgG (for rabbit polyclonal antibodies) or anti-goat IgG (for goat polyclonal antibodies) depending on the primary antibody host species

• Specialized protocols:

  • For ubiquitination studies, include proteasome inhibitors in lysate preparation

  • For interaction studies, consider native gel conditions to preserve protein-protein interactions

How can I optimize co-immunoprecipitation studies involving TRIM54?

For successful co-immunoprecipitation (Co-IP) of TRIM54 and its binding partners:

• Experimental design:

  • Tag strategies: Flag-tag for TRIM54 and Myc-tag for potential binding partners (like YOD1) have been validated

  • Expression system: HEK 293T cells have been successfully used for co-transfection and IP studies

• Protocol optimization:

  • For studying TRIM54-YOD1 interaction:

    • Perform bidirectional Co-IP (IP with TRIM54 antibody and blot for YOD1, then IP with YOD1 antibody and blot for TRIM54)

    • Use tagged proteins for confirmation (e.g., Flag-TRIM54 and Myc-YOD1)

• Controls and validation:

  • Include IgG control to assess non-specific binding

  • Input samples to confirm expression of both proteins

  • For ubiquitination studies, include proteasome inhibitors and ubiquitin immunoblotting

Studies have successfully demonstrated TRIM54 interaction with YOD1 and Axin1 using these approaches .

What experimental approaches can be used to study TRIM54 in muscle disorders?

Given TRIM54's specific expression in heart and skeletal muscle :

• Expression analysis:

  • Compare TRIM54 expression levels in normal vs. diseased muscle tissues

  • Assess localization changes using immunofluorescence

• Functional assessment:

  • Evaluate effects of TRIM54 knockdown/overexpression on:

    • Myoblast differentiation

    • Myotube formation

    • Microtubule stabilization

  • Measure differentiation markers in response to TRIM54 modulation

• Interaction studies:

  • Identify muscle-specific binding partners of TRIM54

  • Determine if these interactions are altered in disease states

• In vivo models:

  • Generate tissue-specific TRIM54 knockout or transgenic mice

  • Analyze muscle phenotypes and function

How can I investigate TRIM54's role in tendon healing and regeneration?

Based on research showing TRIM54's importance in tendinopathy :

• In vitro experimental approach:

  • Isolate and culture tendon-derived stem cells (TDSCs)

  • Create inflammation model using TNF-α exposure

  • Modulate TRIM54 expression via overexpression or knockdown

  • Assess:

    • Stemness (CD146, Sox2, Oct4)

    • Tenogenic differentiation (COL1, collagen type 3, scleraxis, Tnmd)

    • Inflammation markers (IL-1β, IL-6)

    • Apoptotic markers (Bax, c-caspase-3)

• In vivo model system:

  • Use rat tendon injury model

  • Deliver TRIM54 via appropriate vectors

  • Evaluate:

    • Histopathological score

    • Biomechanical properties (failure load, stiffness, Young's modulus)

• Molecular mechanism investigation:

  • Focus on TRIM54-YOD1 interaction

  • Assess ubiquitination patterns

  • Study downstream signaling effects

Research has shown that TRIM54 overexpression improves histopathological scores and biomechanical properties in tendon injury models, suggesting therapeutic potential .

What are the emerging roles of TRIM54 in different disease contexts?

Recent research has revealed TRIM54's involvement in multiple pathological conditions:

What methodological advances would improve TRIM54 research?

To advance understanding of TRIM54 biology:

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize TRIM54 subcellular localization

  • Live-cell imaging to monitor TRIM54 dynamics during myotube formation

• Protein interaction mapping:

  • Proximity labeling techniques (BioID, APEX) to identify TRIM54 interactome

  • Hydrogen-deuterium exchange mass spectrometry to map interaction domains

• In vivo models:

  • Conditional and tissue-specific TRIM54 knockout models

  • CRISPR/Cas9 gene editing to introduce disease-associated mutations

• Therapeutic approaches:

  • Development of small molecules targeting TRIM54-YOD1 or TRIM54-Axin1 interactions

  • Gene therapy approaches to modulate TRIM54 expression in tendinopathy