TRIM2 Antibody

What is TRIM2 and what key cellular functions does it serve?

TRIM2 is a member of the TRIM protein family with multiple biological functions. Research has demonstrated that TRIM2 is involved in several important cellular processes. In neurological contexts, TRIM2 mutations are associated with Charcot-Marie-Tooth disease (CMTD), indicating a crucial role in neuronal function and maintenance . In cancer biology, TRIM2 appears to be associated with glutamine metabolism in breast cancer, particularly in triple-negative breast cancer (TNBC) . Interestingly, TRIM2 also demonstrates antiviral properties, specifically restricting New World arenavirus (NWA) entry into cells .

The protein contains several characteristic domains including a RING domain (though not all isoforms contain a complete RING domain), B-box domains, coiled-coil domains, and NHL repeat regions. Unlike most TRIM family proteins, TRIM2's antiviral activity appears to be independent of its RING domain, which typically encodes ubiquitin ligase activity . This unique characteristic distinguishes TRIM2 from other members of the TRIM family and may contribute to its specialized functions.

Which TRIM2 antibodies have been validated for research applications?

Several TRIM2 antibodies have been rigorously validated for experimental applications. The monoclonal antibody from Proteintech (catalog number 67342-1-Ig) has been extensively validated and successfully employed for both Western blotting and immunohistochemistry applications . For Western blotting protocols, this antibody has been used at dilutions of 1:1000, while for immunohistochemistry applications, it has been effectively used at 1:500 dilution .

Secondary antibodies successfully paired with TRIM2 primary antibodies include IR Dye 800CW donkey anti-rabbit and 680RD donkey anti-mouse at 1:15,000 dilution for Western blotting applications . These combinations have demonstrated reliable results in detecting TRIM2 across multiple experimental contexts. When selecting a TRIM2 antibody for research, it's essential to consider the specific application (Western blot, IHC, IP, etc.) and whether the antibody has been validated for that particular use in peer-reviewed studies.

How should TRIM2 protein expression be detected in tissue samples?

TRIM2 protein expression in tissue samples is typically detected using immunohistochemistry (IHC). Based on validated protocols, an effective approach includes:

• Tissue processing: Formalin-fixed, paraffin-embedded tissue sections (typically 4 μm thick) mounted on tissue microarrays (TMAs) or as full-face sections provide reliable samples for analysis .

• Antigen retrieval: Heat-induced antigen epitope retrieval in citrate buffer (pH 6.0) for 20 minutes using a microwave oven has been established as an effective method for exposing TRIM2 epitopes .

• Detection system: The Novolink polymer detection system (such as RE7150-K from Leica Biosystems) has been successfully employed in multiple studies .

• Antibody incubation: TRIM2 monoclonal antibody (67342-1-Ig, Proteintech) at 1:500 dilution in antibody diluent at room temperature for 90 minutes has shown consistent and reliable results .

• Controls: Negative controls (omission of the primary antibody) and positive controls (liver tissue, according to manufacturer's datasheet) should be included in every experiment to validate staining specificity .

TRIM2 is observed exclusively in the cytoplasm of invasive tumor cells, with intensity ranging from absent to high. Scoring systems typically categorize expression as negative, low, or high based on staining intensity and percentage of positive cells . This methodology has been successfully employed to evaluate TRIM2 expression in breast cancer tissues and correlate expression with clinical outcomes.

How should TRIM2 antibodies be validated before experimental use?

Comprehensive validation of TRIM2 antibodies is essential for generating reliable and reproducible results. A thorough validation approach should include:

• Western blot analysis:

  • Testing the antibody on multiple cell lines with varying TRIM2 expression levels

  • Including appropriate positive controls (e.g., MCF-7 cells) and negative controls

  • Confirming band size matches the predicted molecular weight (approximately 72-80 kDa for TRIM2)

  • Using a housekeeping protein (such as β-actin at 42 kDa) as loading control

• Antibody specificity assessment:

  • Testing for cross-reactivity with other TRIM family proteins

  • Performing knockdown/knockout validation using siRNA or CRISPR/Cas9 targeting TRIM2

  • For polyclonal antibodies, considering pre-absorption testing with immunizing peptide

• Application-specific validation:

  • For IHC: Testing on positive control tissues (such as liver) as recommended by manufacturer

  • Including technical controls (omission of primary antibody)

  • Evaluating staining pattern consistency with known TRIM2 localization (cytoplasmic)

  • Testing on full-face sections to evaluate heterogeneity before using tissue microarrays

In the literature, TRIM2 antibody validation for Western blotting has been successfully performed using multiple breast cancer cell lines, including ER+ (MCF-7, ZR-75-1, HCC1500) and triple-negative (MDA-MB-231, MDA-MB-436, and MDA-MB-468) cell lines . This approach has demonstrated that the antibody can detect TRIM2 across different cellular contexts with high specificity.

What controls are essential when working with TRIM2 antibodies?

When conducting experiments with TRIM2 antibodies, appropriate controls are critical to ensure the validity and reliability of results:

• For Western blotting:

  • Positive control: Cell lines or tissues known to express TRIM2 (e.g., MCF-7 breast cancer cells)

  • Negative control: Cell lines with TRIM2 knockdown/knockout if available

  • Loading control: Housekeeping protein such as β-actin (detected at approximately 42 kDa)

  • Molecular weight marker: To confirm band size matches expected TRIM2 molecular weight (72-80 kDa)

• For immunohistochemistry:

  • Positive tissue control: Tissue known to express TRIM2 (liver tissue has been used based on manufacturer recommendations)

  • Negative reagent control: Omission of primary antibody while maintaining all other steps

  • Internal positive and negative controls: Tissues or cells within the sample known to be positive or negative for TRIM2

  • Isotype control: Primary antibody replaced with non-specific antibody of the same isotype

• For immunoprecipitation:

  • Input control: Aliquot of pre-IP lysate

  • Negative control: IP performed with non-specific antibody of same isotype

  • Knockout/knockdown control: Lysates from cells lacking TRIM2 expression

  • Reciprocal IP: If studying interaction partners, confirm by IP with antibodies against the partner protein

  • When using anti-TRIM2 antibody for IP experiments, control experiments with tissues/cells lacking TRIM2 are essential, as demonstrated in studies where the antibody did not precipitate proteins when used with brain extracts from TRIM2-knockout mice

• For functional studies:

  • Gain-of-function controls: Cells overexpressing TRIM2

  • Loss-of-function controls: Cells with TRIM2 knockdown or knockout

  • Specificity controls: Experiments with related proteins (e.g., TRIM5α has been used as a control to demonstrate specificity of TRIM2's antiviral effects)

These controls help ensure that observed results are specific to TRIM2 and not due to technical artifacts or non-specific antibody interactions.

How does TRIM2 expression correlate with patient outcomes in cancer research?

TRIM2 expression demonstrates significant correlations with patient prognosis, particularly in breast cancer. The research reveals:

• Prognostic significance in triple-negative breast cancer (TNBC):

  • High TRIM2 protein expression is a strong predictor of poor outcomes in TNBC and ER-negative patients

  • Significantly impacts breast cancer-specific survival (BCSS) (p < 0.05)

  • Reduces disease-free survival (DFS) (p < 0.05)

  • Associated with worse distant metastasis-free survival (DMFS) (p < 0.01)

• Association with chemotherapy response:

  • TRIM2 expression holds predictive power for DMFS in all patients receiving chemotherapy (p < 0.05)

  • Particularly strong predictor in TNBC patients receiving chemotherapy (p < 0.05)

  • Strong predictor in ER-negative tumors treated with chemotherapy (p < 0.01)

• Multivariate analysis findings:

  • High TRIM2 protein expression remains an independent predictor of poor outcomes in TNBC and ER-negative tumors

  • Retains significance even after controlling for established prognostic factors including tumor size, tumor grade, and lymph node stage

• Subtype-specific effects:

  • No significant association between TRIM2 expression and patient outcome was observed in ER-positive tumors and non-TNBC

  • This suggests TRIM2's prognostic value is specific to TNBC and ER-negative breast cancers

These findings indicate that TRIM2 could serve as a valuable prognostic biomarker specifically for TNBC patients, with particular relevance for those undergoing chemotherapy. The molecular mechanisms underlying these associations continue to be explored, but may relate to TRIM2's involvement in glutamine metabolism pathways.

What is the relationship between TRIM2 and glutamine metabolism in cancer?

The relationship between TRIM2 and glutamine metabolism in cancer, particularly breast cancer, represents an emerging area of investigation:

• Co-expression with glutaminase (GLS):

  • Pathway analysis identified TRIM2 expression as an important gene co-expressed with high GLS expression in breast cancer

  • GLS is a key enzyme in glutamine metabolism, converting glutamine to glutamate, which is critical for cancer cell metabolism

• Association with triple-negative breast cancer (TNBC):

  • Both high TRIM2 mRNA and protein expression are significantly associated with TNBC (p < 0.01)

  • TNBC is known to be highly dependent on glutamine metabolism for survival and proliferation

• Potential metabolic implications:

  • The co-expression of TRIM2 with GLS suggests TRIM2 may be involved in regulating glutamine metabolism

  • This could explain why TRIM2 overexpression is associated with more aggressive disease and poorer outcomes in TNBC

  • Glutamine serves as an alternative energy source for cancer cells, particularly when glucose availability is limited

• Research gaps:

  • The exact molecular mechanisms linking TRIM2 to glutamine metabolism remain to be fully characterized

  • Research has noted that "the molecular mechanisms and functional behaviour of TRIM2 and the functional link with GLS in BC warrant further exploration using in vitro models"

Understanding the relationship between TRIM2 and glutamine metabolism could provide insights into the aggressive nature of TNBC and potentially reveal new therapeutic approaches for this difficult-to-treat breast cancer subtype. TRIM2 antibodies serve as essential tools in investigating these metabolic connections.

How can TRIM2 antibodies be used to study viral infection mechanisms?

TRIM2 antibodies provide valuable tools for investigating viral infection mechanisms, particularly for New World arenaviruses (NWAs), where TRIM2 has been demonstrated to restrict viral entry:

• Detecting TRIM2 expression changes during infection:

  • Western blotting with TRIM2 antibodies can monitor expression levels before and after viral infection

  • This can help determine if viruses modulate TRIM2 expression as a counter-defense mechanism

  • Immunofluorescence microscopy can examine changes in TRIM2 subcellular localization during infection

• Studying TRIM2's role in virus entry pathways:

  • TRIM2 has been shown to block NWA entry into cells

  • Antibodies can be used to track co-localization of TRIM2 with viral particles during early infection stages

  • Immunoprecipitation with TRIM2 antibodies followed by mass spectrometry can identify interaction partners involved in antiviral activity

• Investigating virus-host protein interactions:

  • Co-immunoprecipitation using TRIM2 antibodies can pull down viral proteins that may interact with TRIM2

  • Proximity ligation assays can detect close associations between TRIM2 and viral components in situ

  • These approaches can help identify potential viral antagonists of TRIM2's antiviral function

• Exploring TRIM2 domains critical for antiviral activity:

  • Studies using TRIM2 deletion mutants have shown that its antiviral activity is uniquely independent of the RING domain

  • TRIM2 antibodies targeting different domains can help identify which regions are accessible during viral infection

• Examining TRIM2's role in phagocytosis pathways:

  • TRIM2 interacts with SIRPA, a known inhibitor of phagocytosis

  • TRIM2 antibodies can be used to study how TRIM2 influences phagocytosis of viral particles

  • This approach has revealed that "NWA entry and phagocytosis pathways overlap"

These applications of TRIM2 antibodies provide valuable insights into the mechanisms of viral restriction and may inform the development of antiviral strategies targeting the TRIM2 pathway.

How can researchers address TRIM2 antibody specificity issues?

When encountering TRIM2 antibody specificity challenges, researchers should implement several strategies to ensure reliable detection:

• Validate antibody specificity:

  • Perform Western blot analysis on samples from TRIM2 knockout/knockdown models

  • Test the antibody on cell lines with varying TRIM2 expression levels

  • Look for the expected band sizes (approximately 72-80 kDa for TRIM2)

  • Verify that the antibody doesn't cross-react with other TRIM family proteins

• Optimize blocking conditions:

  • Test different blocking reagents (BSA, non-fat dry milk, commercial blockers)

  • Adjust blocking time and temperature

  • Increase blocking reagent concentration if non-specific binding is observed

• Modify antibody incubation parameters:

  • Titrate antibody concentration to find optimal signal-to-noise ratio

  • Test different antibody diluents (some may contain additives that reduce background)

  • Adjust incubation time and temperature (longer at 4°C vs. shorter at room temperature)

• Address epitope-specific issues:

  • If multiple isoforms exist, confirm which epitope your antibody recognizes

  • Western blot analysis has shown two bands at the predicted size of approximately 72 and 80 kDa in MCF-7 cells, but just the 72 kDa band in other cell lines

  • Consider testing antibodies targeting different TRIM2 epitopes

• Control experiments:

  • Include technical negative controls (omitting primary antibody)

  • Use tissues/cells known to lack TRIM2 expression as biological negative controls

  • For immunoprecipitation, verify specificity by showing the antibody doesn't precipitate proteins from TRIM2-knockout samples

By systematically addressing these aspects, researchers can significantly improve TRIM2 antibody specificity and generate more reliable experimental results across different applications.

What factors affect TRIM2 detection in experimental settings?

Several factors can influence TRIM2 detection across different experimental settings, potentially leading to variable results:

• Expression level variations:

  • TRIM2 expression varies significantly across tissue and cell types

  • In breast cancer, TRIM2 shows higher expression in TNBC compared to other subtypes

  • Expression levels may change under different experimental conditions (stress, treatments, etc.)

• Sample preparation factors:

  • For tissue samples: Fixation type, duration, and processing methods affect antigen preservation

  • For cell lysates: Lysis buffer composition can impact protein extraction efficiency

  • For IHC: Antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 has been effective)

• Protein isoforms and modifications:

  • Multiple TRIM2 isoforms exist (bands at 72 kDa and 80 kDa have been detected)

  • Alternative splicing can generate protein variants that may or may not be recognized by all antibodies

  • Post-translational modifications may mask antibody epitopes

• Antibody characteristics:

  • Epitope location: Antibodies targeting different regions of TRIM2 may have varying access to the epitope

  • Antibody format: Monoclonal vs. polyclonal antibodies offer different specificity/sensitivity profiles

  • Antibody quality: Lot-to-lot variations can occur, particularly with polyclonal antibodies

• Experimental conditions:

  • Antibody dilution: The optimal dilution varies by application (1:1000 for WB, 1:500 for IHC)

  • Incubation time and temperature affect binding kinetics

  • Secondary antibody selection impacts signal amplification

Understanding these variables and standardizing protocols can help reduce variability and improve reproducibility when detecting TRIM2 across different experimental settings.

What are the key TRIM2 interacting partners that can be co-immunoprecipitated?

TRIM2 antibodies have been used to identify several interacting partners through co-immunoprecipitation studies, revealing insights into TRIM2's functional networks:

• Signal Regulatory Protein α (SIRPA):

  • SIRPA has been identified as a key interaction partner of TRIM2

  • This interaction appears functionally significant as SIRPA also restricts New World arenavirus infection

  • SIRPA is a well-known inhibitor of phagocytosis, suggesting a mechanistic link between TRIM2's antiviral activity and phagocytosis regulation

  • SIRPA's cytoplasmic domain contains tyrosine motifs that, when phosphorylated, become binding sites for SHP-1 and SHP-2, initiating a cascade that blocks phagocytosis

• Verification of co-immunoprecipitation specificity:

  • Control experiments have shown that when anti-TRIM2 antibody is used with brain extracts from TRIM2-knockout mice, no proteins are precipitated, confirming the specificity of the co-immunoprecipitation

  • This control is critical for validating true interaction partners versus non-specific binding

• Potential cancer-related interaction partners:

  • Given TRIM2's association with glutamine metabolism in cancer, proteins involved in this pathway may interact with TRIM2

  • Glutaminase (GLS) expression correlates with TRIM2 expression in breast cancer, though direct interaction has not been confirmed

  • In ER-positive breast cancer studies, TRIM2 has been linked with tamoxifen resistance by mediating apoptosis

  • In ER-negative, basal-like breast cancer, TRIM2 expression is associated with the TNBC-associated nuclear transcription factor SOX10

Future co-immunoprecipitation studies using TRIM2 antibodies, coupled with mass spectrometry, may reveal additional interaction partners and further elucidate TRIM2's role in various cellular processes, from antiviral defense to cancer progression.

What are common pitfalls when using TRIM2 antibodies in co-immunoprecipitation studies?

Co-immunoprecipitation (co-IP) with TRIM2 antibodies presents several challenges that researchers should address to avoid misinterpretation of results:

• Non-specific binding issues:

  • TRIM proteins contain multiple domains that can interact non-specifically

  • Always include appropriate negative controls, such as IgG isotype controls

  • Validate specificity by showing the antibody doesn't precipitate proteins from TRIM2-knockout samples

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Buffer composition challenges:

  • Lysis buffer composition critically affects protein-protein interactions

  • Too stringent: may disrupt genuine interactions (especially weak or transient ones)

  • Too mild: may preserve non-specific interactions

  • Test different detergent types and concentrations (NP-40, Triton X-100, CHAPS)

• Epitope masking:

  • Interaction partners may block the antibody's access to its epitope on TRIM2

  • This can lead to false negatives (failure to immunoprecipitate complexes)

  • Try antibodies targeting different TRIM2 epitopes

  • Consider native vs. denatured IP approaches

• Cross-reactivity with other TRIM proteins:

  • The TRIM family has many members with structural similarities

  • Verify antibody specificity against other TRIM proteins, especially closely related ones

  • Use mass spectrometry to identify all proteins in the immunoprecipitate

• Confirmation strategies:

  • Perform reciprocal IP with antibodies against the interacting partner

  • Use alternative methods to confirm interactions (proximity ligation assay, FRET)

  • Validate functional relevance of interactions through mutation or domain deletion studies

  • For key interactions like TRIM2-SIRPA , confirm with multiple methodologies

By addressing these potential pitfalls, researchers can increase the reliability and significance of TRIM2 co-immunoprecipitation studies, leading to more accurate characterization of TRIM2's interactome and cellular functions.

How to interpret contradictory TRIM2 expression patterns across different studies?

When faced with contradictory TRIM2 expression patterns across different studies, researchers should consider several factors that may explain these discrepancies:

• Methodological differences:

  • Detection techniques vary in sensitivity and specificity (Western blot vs. IHC vs. RNA-seq)

  • Antibody selection: Different antibodies may recognize different epitopes or isoforms

  • Scoring systems: Various IHC scoring methods may use different cutoffs for "high" vs. "low" expression

  • Sample preparation: Differences in fixation, antigen retrieval, or cell lysis protocols

• Biological heterogeneity:

  • TRIM2 expression varies across tissue types and cancer subtypes

  • High TRIM2 expression is associated with TNBC but shows a curious association with low tumor grade

  • Expression may change during disease progression or treatment

  • Intratumoral heterogeneity may lead to sampling bias

• Context-dependent functions:

  • TRIM2 may have different roles in different tissues or cellular contexts

  • In neurological contexts, TRIM2 is associated with Charcot-Marie-Tooth disease

  • In cancer, it's linked to glutamine metabolism and poor outcomes in TNBC

  • In viral infections, it restricts New World arenavirus entry

• Resolution strategies:

  • Perform meta-analyses of available data with standardized criteria

  • Design validation studies that directly address contradictions

  • Use multiple detection methods in the same sample set

  • Conduct functional studies to elucidate context-dependent roles

When interpreting contradictory findings, it's important to recognize that these discrepancies often reflect the complexity of TRIM2 biology rather than errors in individual studies. Understanding the contextual factors and methodological differences can help researchers develop a more nuanced view of TRIM2's roles across different biological systems.