TRIM72 Antibody

What is TRIM72 and what cellular functions does it perform?

TRIM72 is a member of the TRIM superfamily and functions as a RING E3 ubiquitin ligase. It plays critical roles in multiple cellular processes, with its primary function being membrane damage repair through membrane fusion and exocytosis mechanisms. Beyond membrane repair, TRIM72 participates in innate immune responses, insulin resistance regulation, and muscle differentiation processes . The protein contains several functional domains including RING, B-box, coiled-coil, and SPRY domains that mediate its various activities. The RING domain is particularly important for its E3 ligase activity, while the B-box domain facilitates oligomerization that is essential for its membrane repair function .

In which tissues is TRIM72 primarily expressed?

TRIM72 is predominantly expressed in striated muscle tissues but has also been detected in other tissues. Immunoblot analyses consistently show high expression in skeletal muscle and heart tissues from both mouse and rat models . Additionally, TRIM72 has been identified in lung epithelial cells, particularly in alveolar type I (ATI) and alveolar type II (ATII) cells . Immunostaining of lung sections has confirmed the presence of TRIM72 in both T1α-positive ATI cells and surfactant-associated protein C (SP-C)-positive ATII cells . The presence of TRIM72 in these non-muscle tissues explains its protective role in lung and kidney injuries.

What applications are recommended for TRIM72 antibodies in research?

TRIM72 antibodies have been validated for multiple research applications with varying recommended dilutions:

The antibody has been successfully used in knockout/knockdown validation studies, confirming its specificity for TRIM72 . For optimal results, researchers should titrate the antibody concentration for their specific experimental system.

How should TRIM72 antibodies be stored and handled for maximum stability?

For optimal stability and performance, TRIM72 antibodies should be stored at -20°C, where they typically remain stable for one year after shipment. The storage buffer generally consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain antibody integrity . Aliquoting is generally unnecessary for -20°C storage, which simplifies handling procedures. Some antibody preparations may contain 0.1% BSA as a stabilizer, particularly in smaller-volume formats (20μl). When working with the antibody, minimize freeze-thaw cycles and keep on ice while in use. For long-term storage beyond one year, dividing into small aliquots may be beneficial to prevent potential degradation from repeated freeze-thaw cycles.

How does TRIM72 oligomerization impact experimental detection and function?

TRIM72 oligomerization via intermolecular disulfide bonds is a critical aspect of its membrane repair function. During injury, TRIM72 molecules form these intermolecular bonds in response to the oxidative environment, creating oligomers that connect vesicles to the plasma membrane and promote membrane fusion . This oligomerization process has significant implications for experimental detection:

TRIM72 oligomers may appear as higher molecular weight bands on non-reducing SDS-PAGE, which can be distinguished from monomers (53 kDa) by comparing reducing versus non-reducing conditions. For accurate assessment of oligomerization states, researchers should:

• Use freshly prepared samples to prevent artificial oxidation

• Include both reducing and non-reducing conditions in Western blot analyses

• Consider using gradient gels (4-15%) to better resolve high molecular weight oligomers

• Employ crosslinking agents like disuccinimidyl suberate (DSS) to stabilize transient oligomeric states

The crystal structure of the TRIM72 BCC-SPRY domains has revealed that the B-box domain dimerization facilitates TRIM72 oligomerization, providing key structural insights into this process . Mutations affecting key residues in the B-box domain can disrupt oligomerization and consequently impair membrane repair function, making these residues important targets for structure-function studies.

What are the optimal methods for studying TRIM72-lipid interactions?

TRIM72 interacts with negatively charged phospholipids, particularly phosphatidylserine (PS), which is critical for its membrane localization and repair function. Flow cytometry analysis has shown that TRIM72 interacts with PS-containing liposomes with higher affinity for 30 mol% PS than for 10 mol% PS, suggesting that clustered and negatively charged phospholipid domains are required for effective TRIM72–PS binding .

For researchers investigating TRIM72-lipid interactions, several methodological approaches are recommended:

• Liposome Coflotation Assays: Prepare liposomes with varying compositions of negatively charged phospholipids (particularly PS) to assess binding specificity and affinity. This method can be used with purified TRIM72 or mutant variants to identify key residues involved in lipid binding.

• Lipid Strip Assays: Commercial membrane lipid strips can be used to screen for interactions with multiple lipid types simultaneously. Recent findings indicate that TRIM72 interacts with several kinds of negatively charged lipids beyond just phosphatidylserine .

• Surface Plasmon Resonance (SPR): For quantitative binding kinetics, SPR with immobilized lipid bilayers of defined composition can provide detailed affinity measurements.

• Fluorescence Microscopy with Labeled Lipids: Co-localization studies using fluorescently labeled lipids and TRIM72 can provide spatial information about these interactions in cellular contexts.

When designing these experiments, it's important to control for oxidation state, as TRIM72's lipid-binding properties may be influenced by its oligomerization status.

What controls are essential when using TRIM72 knockout models?

When working with TRIM72 knockout (KO) models, several critical controls should be implemented to ensure experimental validity:

• Genotype Verification: Confirm genotype using established PCR protocols. For TRIM72 KO mice, use the forward primer 5′-CCTTCTGCGTCAGGAACTGTCCTGC-3′ and reverse primer 5′-CAGCAGTCCCACCCTGCCTTCACCG-3′. The null allele generates a 1,250-bp fragment while the wild-type allele produces a 480-bp fragment .

• Protein Expression Validation: Confirm the absence of TRIM72 protein by Western blot analysis using validated antibodies in relevant tissues (skeletal muscle, heart, lung).

• Background Strain Matching: When comparing KO and wild-type animals, ensure they have been backcrossed to the same genetic background for at least 5-10 generations to minimize background effects. TRIM72 KO mice should be crossed to 129/C56BL/6J wild type mice for at least five generations to minimize genetic background discrepancy .

• Age and Sex Matching: Use age-matched (2-6 months) and sex-matched cohorts for all experiments, as TRIM72 expression and function may vary with age and sex.

• Rescue Experiments: Include rescue experiments where TRIM72 expression is restored (e.g., using TRIM72 overexpressor mice) to confirm that phenotypes are specifically due to TRIM72 loss.

For membrane repair studies specifically, incorporate:

• Injury Models: Use established injury models like mechanical ventilation-induced lung injury with propidium iodide (PI) staining to assess cell membrane integrity .

• Quantitative Readouts: Measure cell injury index (e.g., number of PI-positive cells as a fraction of total cells) to quantitatively assess membrane repair capacity.

How can researchers optimize immunoprecipitation protocols for TRIM72 interaction studies?

Immunoprecipitation (IP) is a valuable technique for studying TRIM72 protein interactions, particularly its associations with membrane repair complex components. Based on published protocols, the following optimizations are recommended:

• Lysis Buffer Selection: Use a mild non-ionic detergent (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions. Include protease inhibitors and, importantly, add N-ethylmaleimide (NEM) to prevent post-lysis disulfide bond formation or rearrangement.

• Antibody Amount: Use 0.5-4.0 μg of TRIM72 antibody for 1.0-3.0 mg of total protein lysate . Pre-clear lysates with protein A/G beads before adding the antibody to reduce non-specific binding.

• Incubation Conditions: Conduct antibody incubation overnight at 4°C with gentle rotation to maximize specific binding while minimizing non-specific interactions.

• Washing Stringency: Use a graduated washing approach with decreasing salt concentrations to remove non-specific binding while preserving specific interactions. Generally, 3-5 washes are sufficient.

• Elution Method: For mass spectrometry applications, consider on-bead digestion rather than elution to minimize contaminants. For Western blot analysis, elution with SDS sample buffer is typically sufficient.

• Controls: Always include an isotype control (rabbit IgG for 22151-1-AP antibody) and, when possible, a TRIM72 knockout sample as a negative control.

• Crosslinking Consideration: For transient or weak interactions, consider using a crosslinking agent before lysis to stabilize the complexes.

This protocol has been successfully used to detect TRIM72 in mouse heart tissue samples , but may require modifications for other tissue types or specific experimental questions.

How can TRIM72 antibodies be used to investigate the structural basis of membrane repair?

The crystal structure of TRIM72's BCC-SPRY domains has provided valuable insights into its oligomerization mechanism and membrane repair function . Researchers can leverage TRIM72 antibodies for structure-function investigations through:

• Domain-Specific Antibodies: Develop or obtain antibodies targeting specific domains (RING, B-box, coiled-coil, SPRY) to investigate domain-specific functions in membrane repair. These can be particularly useful in immunofluorescence studies to track domain-specific localization during membrane damage.

• Conformational Antibodies: Generate antibodies that specifically recognize oligomeric versus monomeric TRIM72 states to track the dynamic changes in TRIM72 conformation during the repair process.

• Proximity Ligation Assays (PLA): Combine TRIM72 antibodies with antibodies against potential interaction partners (e.g., dysferlin, caveolin) in PLA to visualize and quantify protein interactions at sites of membrane damage with nanometer resolution.

• Super-Resolution Microscopy: Use fluorescently-labeled TRIM72 antibodies with techniques like STORM or PALM to visualize the nanoscale organization of TRIM72 at membrane repair sites, which can provide insights into how its structural properties facilitate repair.

• Structure-Guided Mutagenesis: Based on the crystal structure, design mutations at key interfaces and use antibodies to track how these mutations affect TRIM72 localization, oligomerization, and function.

When designing these experiments, researchers should consider that antibody binding might potentially interfere with certain protein interactions or conformational changes, so complementary approaches should be used for validation.

What are the challenges in detecting TRIM72 in different tissue types?

While TRIM72 was initially characterized in muscle tissues, it is now known to be present in various other tissues including lung epithelial cells and kidney, presenting unique detection challenges:

• Expression Level Variation: TRIM72 expression levels vary significantly across tissues, being highest in skeletal muscle and heart, and considerably lower in other tissues. This requires optimized extraction protocols and potentially higher antibody concentrations for low-expressing tissues.

• Tissue-Specific Isoforms: There may be tissue-specific isoforms or post-translationally modified variants of TRIM72 that affect antibody recognition. Researchers should validate antibodies specifically for each tissue type of interest.

• Extraction Efficiency: Different tissues require different extraction protocols to efficiently solubilize TRIM72. For muscle tissues, stronger lysis buffers containing ionic detergents may be needed, while epithelial tissues might require gentler conditions.

• Fixation Sensitivity: For immunohistochemistry and immunofluorescence, the accessibility of TRIM72 epitopes may be differently affected by fixation methods depending on the tissue type. For lung tissue, paraformaldehyde fixation at transpulmonary pressure of 20 cmH₂O for 30 min at 4°C has been successful .

• Cross-Reactivity in Complex Tissues: In tissues with complex cellular composition, cross-reactivity with other TRIM family members may occur. Validation using knockout tissues as negative controls is highly recommended.

For lung tissues specifically, immunostaining for TRIM72 should be performed alongside markers for specific cell types, such as T1α for ATI cells and surfactant-associated protein C (SP-C) for ATII cells, to accurately characterize its cellular distribution .

How can researchers quantitatively assess TRIM72's membrane repair function?

Quantitative assessment of TRIM72's membrane repair function is essential for both basic mechanistic studies and therapeutic development. Several methodological approaches have been validated:

• In Situ Lung Injury Assay: This established method involves mechanical ventilation followed by propidium iodide (PI) staining to assess membrane integrity. The number of PI-positive alveolus resident cells is measured as a fraction of the total number of alveoli, defining a cell injury index . This approach can compare wild-type, TRIM72 knockout, and TRIM72 overexpressor models to quantify the impact of TRIM72 on repair capacity.

• Laser Injury Models: Focused laser injury of cell membranes combined with real-time imaging of fluorescent dye entry (e.g., FM1-43 or PI) can provide kinetic measurements of membrane resealing. The rate of fluorescence increase and the plateau level achieved can serve as quantitative metrics of repair efficiency.

• Mechanical Scratch Assays: Cell monolayers can be subjected to standardized mechanical scratches, followed by measurement of cell survival and recovery rates. This approach is particularly useful for comparing different cell types or genetic manipulations.

• Flow Cytometry-Based Assays: Following standardized membrane damage (e.g., saponin treatment), cells can be stained with viability dyes and analyzed by flow cytometry to quantify the percentage of cells that successfully repair their membranes.

• Biochemical Assays: Measuring the release of intracellular enzymes (e.g., lactate dehydrogenase) following membrane damage can provide a biochemical readout of repair capacity that can be easily quantified.

For all these assays, appropriate controls including TRIM72 knockout and overexpression models should be included, and experimental conditions should be standardized to ensure reproducibility.

How can TRIM72 antibodies be used to validate therapeutic approaches targeting membrane repair?

TRIM72 has emerged as a promising therapeutic target for conditions involving membrane damage, including muscular dystrophy, cardiovascular disease, and acute lung and kidney injuries . Researchers can use TRIM72 antibodies to support therapeutic development through:

• Target Engagement Studies: Confirm that therapeutic agents (small molecules, peptides, or biologics) actually engage with TRIM72 using co-immunoprecipitation, thermal shift assays, or cellular co-localization studies with TRIM72 antibodies.

• Mechanism of Action Validation: Determine whether therapeutic candidates affect TRIM72 oligomerization, phospholipid binding, or protein interactions using biochemical assays with TRIM72 antibodies as detection reagents.

• Biodistribution Analysis: Track the distribution of recombinant TRIM72 therapies in animal models using immunohistochemistry or Western blotting with antibodies that can distinguish endogenous from exogenous TRIM72.

• Pharmacodynamic Biomarkers: Develop assays that measure TRIM72 activation status or complex formation as pharmacodynamic biomarkers for clinical trials using specific antibodies.

• Patient Stratification: Investigate whether TRIM72 expression levels or variants correlate with disease severity or therapeutic response, potentially identifying patient subgroups most likely to benefit from TRIM72-targeted therapies.

In these applications, antibody specificity is crucial, and validation should include appropriate controls such as TRIM72 knockout tissues or cells . The commercial antibody 22151-1-AP has been validated for multiple applications including Western blot, immunohistochemistry, and immunoprecipitation, making it versatile for these translational research applications .

What are the considerations for studying TRIM72 in disease models?

When investigating TRIM72 in disease models, several important considerations should guide experimental design:

• Disease-Specific Expression Patterns: TRIM72 expression may be altered in disease states. Baseline characterization of TRIM72 levels and localization in the disease model is essential before intervention studies.

• Timing of Analysis: The dynamic nature of TRIM72's response to membrane damage means that timing of sample collection is critical. For acute injury models, time-course studies with multiple collection points are recommended.

• Oxidative Environment: Since TRIM72 function is intimately linked to oxidation-dependent oligomerization, the oxidative status of the disease model should be characterized and controlled where possible. Consider measuring markers of oxidative stress alongside TRIM72 analyses.

• Compensatory Mechanisms: In chronic disease models, compensatory upregulation of other membrane repair proteins may occur. A comprehensive analysis should include other repair machinery components like dysferlin and annexins.

• Therapeutic Intervention Design: When testing TRIM72-based interventions, consider the timing, dosage, and delivery method carefully. For recombinant TRIM72 therapy, the protein's half-life and biodistribution should be established.

• Knockout Considerations: Complete TRIM72 knockout may have developmental effects that complicate interpretation of disease models. Consider using inducible or tissue-specific knockout approaches or heterozygous models that maintain partial function.

• Translational Relevance: Ensure that the disease model accurately reflects the human condition, particularly regarding TRIM72 expression patterns and regulation, which may differ between species.

In cardiovascular, muscular dystrophy, and acute lung injury models, TRIM72 has shown protective effects, suggesting therapeutic potential that warrants further investigation with carefully designed preclinical studies .

What are common challenges in Western blot detection of TRIM72?

Researchers frequently encounter specific challenges when detecting TRIM72 by Western blot that can be addressed through optimized protocols:

• Multiple Bands: TRIM72 may appear as multiple bands due to post-translational modifications or oligomerization. To differentiate between these possibilities:

  • Compare reducing vs. non-reducing conditions

  • Use phosphatase treatment to identify phosphorylated forms

  • Include denaturing agents like urea to disrupt aggregates

• Weak Signal in Non-Muscle Tissues: Due to lower expression levels in non-muscle tissues, signal enhancement strategies may be necessary:

  • Increase protein loading (up to 50-100 μg for lung or kidney samples)

  • Use high-sensitivity detection reagents (ECL Prime or Femto)

  • Consider longer exposure times while monitoring background

• Non-Specific Bands: To reduce non-specific signals:

  • Increase blocking time (overnight at 4°C)

  • Use 5% BSA instead of milk for blocking

  • Include 0.1% Tween-20 in wash buffers

  • Consider more stringent washing (increase salt concentration)

• Sample Preparation Issues: TRIM72 detection can be sensitive to sample preparation:

  • Add protease inhibitors and keep samples cold

  • Include reducing agents (DTT or β-mercaptoethanol) to prevent artificial oligomerization

  • For membrane-associated TRIM72, ensure complete solubilization with appropriate detergents

• Antibody Dilution Optimization: The recommended dilution range for Western blot is 1:2000-1:10000 , but this should be empirically determined for each tissue type and experimental condition.

Using validated positive controls (mouse skeletal muscle or heart tissue) alongside experimental samples can help troubleshoot these issues and ensure reliable detection.

How can researchers optimize immunofluorescence protocols for TRIM72 localization studies?

Immunofluorescence (IF) is crucial for understanding TRIM72's subcellular localization and dynamics during membrane repair. For optimal results:

• Fixation Method Selection:

  • For muscle tissue: 4% paraformaldehyde for 15-20 minutes at room temperature

  • For lung tissue: 4% paraformaldehyde at transpulmonary pressure of 20 cmH₂O for 30 min at 4°C

  • Avoid methanol fixation which can disrupt membrane structures

• Permeabilization Optimization:

  • Use mild permeabilization (0.1-0.2% Triton X-100 for 5-10 minutes)

  • For membrane studies, consider saponin (0.1%) which preserves membrane structures better than Triton

• Blocking Strategy:

  • Use 5-10% normal serum (species different from primary antibody)

  • Include 1-2% BSA to reduce non-specific binding

  • Consider adding 0.1-0.3% glycine to quench aldehyde groups from fixation

• Antibody Dilution:

  • Start with the recommended 1:400-1:1600 dilution range

  • Optimize by testing a dilution series

  • Incubate primary antibody overnight at 4°C for best signal-to-noise ratio

• Signal Amplification:

  • For tissues with low TRIM72 expression, consider using tyramide signal amplification

  • Biotin-streptavidin systems can also enhance detection sensitivity

• Co-localization Studies:

  • Include markers for specific subcellular structures (plasma membrane, vesicles)

  • For lung tissue, co-stain with T1α for ATI cells and SP-C for ATII cells

  • Use appropriate controls to account for spectral bleed-through

• Image Acquisition:

  • Use confocal microscopy for precise subcellular localization

  • Consider super-resolution techniques for detailed membrane structure analysis

  • Acquire z-stacks to fully capture TRIM72 distribution in three dimensions

These optimizations should be combined with appropriate controls, including TRIM72 knockout tissues and secondary-only controls, to ensure specificity of the observed signals.