TRIM54 Antibody, Biotin conjugated

What is TRIM54 and why is it important in research?

TRIM54 (Tripartite motif-containing protein 54), also known as MURF, MURF3, or RNF30, is a protein primarily expressed in heart and skeletal muscle tissue. It exists in two isoforms with molecular weights of 40kDa and 45kDa and has the ability to form both homooligomers and heterooligomers . TRIM54 plays a critical role in binding and stabilizing microtubules during myotube formation, making it an important target for muscle development research . Recent studies have revealed TRIM54's function in alleviating inflammation and apoptosis by interacting with and stabilizing YOD1 through ubiquitination . The downregulation of TRIM54 in tendinopathy samples suggests its potential therapeutic role in treating musculoskeletal disorders, making it an increasingly important research target .

What applications are TRIM54 antibodies validated for?

TRIM54 antibodies have been validated for multiple applications depending on the specific product. For example, Proteintech's Rabbit Recombinant TRIM54 antibody (83175-2-RR) is validated for Western Blot (WB), Immunofluorescence (IF/ICC), Flow Cytometry (FC), and ELISA applications . The recommended dilutions vary by application:

Biotin-conjugated versions, such as the CSB-PA861180LD01HU from Cusabio, are specifically validated for ELISA applications and offer additional advantages for detection systems .

Why use biotin-conjugated antibodies for TRIM54 detection?

Biotin-conjugated antibodies provide significant advantages in research applications due to the exceptionally strong interaction between biotin and (strept)avidin proteins. This interaction enables enhanced detection sensitivity in various assay formats. Anti-biotin antibodies have been shown to enable unprecedented enrichment of biotinylated peptides from complex mixtures . When used in protocols like live-cell proximity labeling with APEX peroxidase, anti-biotin enrichment followed by mass spectrometry can yield over 1,600 biotinylation sites on hundreds of proteins—an increase of more than 30-fold compared to streptavidin-based enrichment of proteins . For TRIM54 research, biotin conjugation allows for flexible detection strategies and can be particularly valuable when studying protein-protein interactions or when enhanced sensitivity is required.

How should TRIM54 antibody, biotin conjugated be stored and handled?

Proper storage and handling are critical for maintaining antibody activity. For biotin-conjugated TRIM54 antibodies:

• Store at -20°C or -80°C upon receipt .

• Avoid repeated freeze-thaw cycles, which can degrade antibody quality and performance .

• The antibody is typically supplied in a buffer containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) .

• For working aliquots, smaller volumes can be prepared and stored according to the manufacturer's recommendations to minimize freeze-thaw cycles.

• Prior to use, thaw completely and mix gently to ensure homogeneity.

These storage conditions help maintain the integrity of both the antibody and its biotin conjugation for optimal research results.

How can TRIM54 antibody, biotin conjugated be used to study TRIM54-YOD1 interactions?

Recent research has revealed that TRIM54 interacts with and ubiquitinates YOD1, a deubiquitinating enzyme of the ovarian tumor family . To investigate this interaction using biotin-conjugated TRIM54 antibodies, researchers can implement the following methodology:

• Co-immunoprecipitation (Co-IP): Use the biotin-conjugated TRIM54 antibody with streptavidin beads to capture TRIM54 and its interacting partners. This approach has been validated in studies where TRIM54 was shown to bind YOD1 .

• Ubiquitination Assays: After overexpression of TRIM54, researchers observed increased ubiquitination of endogenous YOD1 in HEK 293T cells. These experiments revealed that TRIM54 significantly increases HA-tagged ubiquitin levels in cells co-expressing Myc-tagged YOD1 .

• Cycloheximide Chase Assay: This method was used to illustrate the relationship between TRIM54 and YOD1, showing that YOD1 levels decreased with increasing exposure to cycloheximide, an effect that was enhanced in cells overexpressing TRIM54 .

• Reciprocal Co-IP: When proteins immunoprecipitated using TRIM54 antibodies were assessed by Western blot, YOD1 protein was detected. Similarly, when proteins were immunoprecipitated with YOD1 antibody, TRIM54 was detected .

For these experiments, the biotin-conjugated antibody provides an advantage in pull-down assays where streptavidin matrices can be used for efficient capture of the TRIM54-YOD1 complex.

What experimental considerations are important when using TRIM54 antibody, biotin conjugated in tendinopathy research?

When utilizing biotin-conjugated TRIM54 antibodies in tendinopathy research, several important experimental considerations should be addressed:

• Tissue-specific expression variations: TRIM54 levels are significantly downregulated in human tendinopathy samples compared to normal tendons . This differential expression must be considered when designing experiments and interpreting results.

• Model systems: Research has employed both in vitro models with tendon-derived stem cells (TDSCs) and in vivo rat tendon injury models . For in vitro studies, TDSCs exposed to TNF-α exhibited decreased TRIM54 expression, reduced differentiation potential, increased inflammatory markers, and increased apoptosis .

• Overexpression studies: When investigating therapeutic potential, TRIM54 overexpression significantly rescued impaired TDSCs, improving proliferation and differentiation . Biotin-conjugated antibodies can be used to confirm overexpression and track protein localization.

• Functional readouts: Important parameters to measure include:

  • Stemness markers (CD146, Sox2, Oct4)

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

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

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

• In vivo assessment: TRIM54 overexpression significantly improved histopathology and mechanical properties (failure load, stiffness, Young's modulus) in tendon injury animal models .

The biotin-conjugated antibody allows for versatile detection in these various experimental systems, particularly for immunohistochemistry and protein quantification assays.

How can researchers optimize TRIM54 antibody, biotin conjugated for proximity labeling experiments?

Proximity labeling has emerged as a powerful technique for identifying protein-protein interactions in living cells. For optimizing TRIM54 antibody, biotin conjugated in proximity labeling experiments:

• Selection of proximity labeling system: APEX peroxidase-based systems have demonstrated success with biotin labeling, yielding over 1,600 biotinylation sites when combined with anti-biotin enrichment and mass spectrometry .

• Anti-biotin enrichment strategy: Anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex peptide mixtures, offering a 30-fold increase in identification of biotinylation sites compared to streptavidin-based enrichment .

• Sample preparation considerations:

  • For heart and skeletal muscle tissues, where TRIM54 is primarily expressed, special attention to tissue lysis and protein extraction is necessary .

  • For cellular models, C2C12 and HepG2 cells have demonstrated positive IF/ICC detection .

• Validation in specific cell types: Consider testing in cell lines where TRIM54 has been confirmed, such as MCF-7 cells (positive for FC), C2C12 cells, and HepG2 cells (positive for IF/ICC) .

• Controls: Include appropriate controls to distinguish specific from non-specific biotinylation, such as samples without the proximity labeling enzyme or with a catalytically inactive mutant.

This approach can be particularly valuable for identifying novel interaction partners of TRIM54 beyond the already established YOD1 interaction .

What are the technical challenges in detecting TRIM54 in different tissue samples and how can they be addressed?

Detecting TRIM54 in different tissue samples presents several technical challenges that researchers should be aware of:

• Tissue-specific expression patterns: TRIM54 is primarily expressed in heart and skeletal muscle tissues . Detection in other tissues may require more sensitive methods or may not be feasible depending on expression levels.

• Sample-dependent optimization: The manufacturer notes that dilution requirements can be sample-dependent, necessitating optimization for each tissue type or experimental system .

• Reactivity considerations: Confirmed reactivity with human and mouse samples has been established , but testing in other species may require validation.

• Detection of multiple isoforms: TRIM54 exists in two isoforms with molecular weights of 40kDa and 45kDa , which may need to be distinguished in experimental analyses.

• Addressing challenges:

  • For low-expressing samples, consider using amplification systems that leverage the biotin-streptavidin interaction

  • For muscle tissues, optimize protein extraction protocols for fibrous tissues

  • Use appropriate positive controls (human heart tissue, mouse heart tissue) and negative controls

  • When analyzing Western blots, be aware of the expected molecular weight (40 kDa) and potential isoform variations

  • Consider microscopy-based detection for spatial information, as TRIM54 antibodies have been validated for immunofluorescence in specific cell types

These considerations are particularly important when using biotin-conjugated antibodies, as the conjugation may affect antibody performance in certain applications.

How can TRIM54 antibody, biotin conjugated be used to investigate the role of TRIM54 in muscle development and disease?

TRIM54 antibody, biotin conjugated offers several methodological approaches for investigating the role of TRIM54 in muscle development and disease:

• Developmental studies: Since TRIM54 binds and stabilizes microtubules during myotube formation , developmental time-course experiments can be designed to track TRIM54 expression and localization during myogenesis. Using biotin-conjugated antibodies allows for multiplexed imaging with other developmental markers.

• Disease model investigations: In tendinopathy and other muscular disorders where TRIM54 is downregulated , the antibody can be used to:

  • Quantify protein expression levels in patient samples

  • Validate animal models by confirming reduced TRIM54 expression

  • Track therapeutic interventions aimed at restoring TRIM54 levels

• Mechanistic studies: To understand TRIM54's role in alleviating inflammation and apoptosis:

  • Examine TRIM54-YOD1 interactions in normal vs. diseased muscle tissue

  • Track changes in ubiquitination patterns using the biotin-conjugated antibody in pull-down assays

  • Study downstream effectors affected by TRIM54, such as inflammatory markers (IL-1β, IL-6) and apoptotic markers (Bax, c-caspase-3)

• Therapeutic development: For developing TRIM54-based therapies:

  • Screen compounds that upregulate TRIM54 expression

  • Validate gene therapy approaches using the antibody to confirm protein expression

  • Monitor improvements in histopathological features and mechanical properties of tendons following treatments

The biotin conjugation offers advantages in these applications through enhanced detection sensitivity and compatibility with streptavidin-based imaging or purification systems.

How can researchers troubleshoot non-specific binding when using TRIM54 antibody, biotin conjugated?

Non-specific binding is a common challenge when working with antibodies. For biotin-conjugated TRIM54 antibodies, consider these troubleshooting approaches:

• Optimize blocking conditions:

  • Use protein-free blockers when possible to reduce background

  • Consider specific blocking of endogenous biotin using avidin/biotin blocking kits

  • Test different blocking agents (BSA, normal serum, commercial blockers)

• Titrate antibody concentration:

  • Start with recommended dilutions (e.g., 1:2000-1:14000 for WB, 1:50-1:500 for IF/ICC)

  • Perform serial dilutions to determine optimal concentration

  • Remember that biotin conjugation may alter optimal concentration compared to unconjugated antibodies

• Validate specificity:

  • Include proper negative controls (samples not expressing TRIM54)

  • Use positive controls (human heart tissue, mouse heart tissue)

  • Consider TRIM54 knockdown/knockout samples as definitive controls

• Address endogenous biotin issues:

  • Tissues like liver, kidney, and brain have high endogenous biotin

  • Pre-block with avidin or streptavidin before adding the biotin-conjugated antibody

  • Consider alternative detection methods if endogenous biotin is problematic

• Modify washing conditions:

  • Increase washing duration or number of washes

  • Adjust detergent concentration in wash buffers

  • Use high-salt washes for high-background samples

These approaches should be systematically tested to determine the optimal conditions for specific detection of TRIM54 in your experimental system.

What are the best practices for validating experimental results using TRIM54 antibody, biotin conjugated?

Proper validation is critical for ensuring reliable results with TRIM54 antibody, biotin conjugated:

• Multi-technique validation:

  • Confirm findings using different detection methods (WB, IF/ICC, FC, ELISA)

  • Corroborate protein-level findings with mRNA expression data

  • Use multiple antibody clones targeting different epitopes when possible

• Controls for biotin-streptavidin system:

  • Include controls for endogenous biotin

  • Test detection system components separately

  • Consider including biotinylated control proteins

• Genetic validation:

  • Use TRIM54 knockdown/knockout samples

  • Perform rescue experiments with TRIM54 overexpression

  • Consider tag-based detection as an orthogonal approach

• Application-specific validations:

  • For Co-IP studies: validate with reciprocal IP and include IgG controls

  • For ubiquitination studies: include controls with deubiquitinases

  • For proximity labeling: include spatial and temporal controls

• Replication across experimental models:

  • Test in multiple cell lines where TRIM54 is expressed

  • Validate findings in primary cells and tissues

  • Consider both in vitro and in vivo models when possible

Following these validation practices will strengthen research findings and improve reproducibility when working with TRIM54 antibody, biotin conjugated.

How can TRIM54 antibody, biotin conjugated be used in multiplexed imaging studies?

Multiplexed imaging allows visualization of multiple targets simultaneously. Biotin-conjugated TRIM54 antibodies offer several advantages in this context:

• Flexible detection systems:

  • Can be detected with various streptavidin conjugates (fluorophores, quantum dots, enzymes)

  • Compatible with tyramide signal amplification for enhanced sensitivity

  • Can be used in sequential staining protocols

• Optimal combinations:

  • Pair with antibodies against known TRIM54 interaction partners like YOD1

  • Combine with markers for muscle differentiation (e.g., myogenin, MyoD)

  • Co-stain with markers for inflammation in tendinopathy studies

• Implementation strategies:

  • For IF/ICC, use recommended dilutions (1:50-1:500) as starting points

  • When combining with other biotin-conjugated antibodies, employ sequential detection with blocking steps

  • For tissue sections, consider antigen retrieval optimization for muscle tissues

• Data analysis considerations:

  • Account for potential bleed-through between channels

  • Use proper controls for autofluorescence in muscle tissue

  • Employ spectral unmixing for closely overlapping fluorophores

Multiplexed imaging provides spatial context for TRIM54 interactions and localization, enhancing understanding of its role in muscle development and pathology.

What methodological approaches can be used to study TRIM54's ubiquitination activity using biotin-conjugated antibodies?

TRIM54's role as an E3 ubiquitin ligase that targets YOD1 can be studied using several methodological approaches with biotin-conjugated antibodies:

• Ubiquitination assays:

  • Co-transfect cells with tagged TRIM54, YOD1, and ubiquitin constructs

  • Use biotin-conjugated TRIM54 antibody to immunoprecipitate TRIM54 complexes

  • Detect ubiquitinated species using anti-ubiquitin antibodies

  • This approach successfully demonstrated that TRIM54 overexpression increased ubiquitination of endogenous YOD1

• Cycloheximide chase experiments:

  • Treat cells with cycloheximide to inhibit protein synthesis

  • Track YOD1 degradation over time in the presence and absence of TRIM54

  • Use biotin-conjugated TRIM54 antibody to confirm TRIM54 expression

  • This method revealed accelerated YOD1 degradation in cells overexpressing TRIM54

• Deubiquitinase competition assays:

  • Perform co-immunoprecipitation in the presence of deubiquitinase 3

  • Observe how deubiquitinase activity counteracts TRIM54-mediated ubiquitination

  • Use biotin-conjugated TRIM54 antibody to confirm consistent TRIM54 levels

• Domain mapping experiments:

  • Create TRIM54 mutants lacking specific functional domains

  • Test their ability to bind and ubiquitinate YOD1

  • Use biotin-conjugated antibodies against preserved epitopes to detect mutant proteins

These approaches provide complementary evidence for TRIM54's ubiquitin ligase activity and its specific targeting of YOD1, helping to elucidate the mechanism by which TRIM54 reduces inflammation in tendinopathy.

What are emerging applications for TRIM54 antibody, biotin conjugated in regenerative medicine research?

TRIM54 antibody, biotin conjugated has significant potential in regenerative medicine research, particularly for musculoskeletal disorders:

• Therapeutic development monitoring:

  • Track TRIM54 expression in response to potential therapeutic compounds

  • Monitor restoration of normal TRIM54 levels in disease models

  • Assess downstream effects on inflammation and apoptosis markers

• Stem cell differentiation studies:

  • Monitor TRIM54 expression during differentiation of stem cells into muscle lineages

  • Track effects of TRIM54 overexpression on stemness markers (CD146, Sox2, Oct4) and tenogenic differentiation

  • Evaluate TRIM54's role in sphere formation and cell migration during tissue repair

• Tissue engineering applications:

  • Use as a quality control marker for engineered muscle tissues

  • Assess TRIM54 expression in bioengineered tendons as a metric of proper development

  • Monitor mechanical properties in relation to TRIM54 expression levels

• Translational biomarker development:

  • Develop diagnostic tests for tendinopathy based on TRIM54 expression

  • Create prognostic markers for muscle repair based on TRIM54 levels

  • Develop companion diagnostics for TRIM54-targeted therapies

The biotin conjugation provides flexibility in detection methods across these applications, allowing for both sensitive detection and potential therapeutic targeting strategies.

How might TRIM54 antibody, biotin conjugated research contribute to understanding other TRIM family proteins?

Research using TRIM54 antibody, biotin conjugated can provide valuable insights into the broader TRIM protein family:

• Comparative studies across TRIM family members:

  • Several TRIM family members (TRIM11, TRIM16, TRIM21, TRIM27, TRIM54, TRIM55, TRIM63, TRIM71) have been studied in tendinopathy

  • TRIM11, TRIM27, TRIM54, TRIM55, and TRIM63 are downregulated in tendinopathic tendons, while TRIM21 is upregulated

  • Comparative analysis can reveal shared and unique functions

• Ubiquitination mechanism investigations:

  • TRIM proteins share a common tripartite motif structure

  • Insights from TRIM54's ubiquitination of YOD1 may inform understanding of substrate recognition by other TRIM proteins

  • Biotin-conjugated antibodies facilitate detailed interaction studies

• Tissue-specific expression patterns:

  • TRIM54 is primarily expressed in heart and skeletal muscle

  • Understanding tissue-specificity determinants may inform therapeutic targeting of other TRIM proteins

  • Multiplexed studies with biotin-conjugated antibodies can map expression patterns

• Structure-function relationships:

  • TRIM54 can form homooligomers and heterooligomers

  • Investigating oligomerization patterns may provide insights applicable to other TRIM proteins

  • Biotin-conjugated antibodies can be used in proximity labeling to capture transient interactions