TRIM11 Antibody

What is TRIM11 and what are its primary biological functions?

TRIM11 (Tripartite motif-containing protein 11) is an E3 ubiquitin-protein ligase that promotes the degradation of insoluble ubiquitinated proteins. It mediates the ubiquitination of various target proteins, leading to their proteasomal degradation . TRIM11 plays crucial roles in:

TRIM11 mechanistically binds to both the proteasome and USP14 (a deubiquitinase), preventing their association and thereby increasing proteasome activity .

What applications can TRIM11 antibodies be used for in research?

TRIM11 antibodies have been validated for multiple experimental applications:

For optimal results, it is recommended to titrate the antibody concentration in each specific experimental system .

What is the molecular weight of TRIM11 and how does this impact antibody detection?

The calculated molecular weight of TRIM11 is approximately 52 kDa, while the observed molecular weight in SDS-PAGE is typically around 53 kDa . This slight discrepancy is common for many proteins due to post-translational modifications or the inherent properties of the protein. When performing Western blot analysis, researchers should expect to detect a band at approximately 53 kDa when using anti-TRIM11 antibodies. Always include appropriate positive controls (e.g., lysates from HEK-293, HeLa, or Jurkat cells) which have been verified to express detectable levels of TRIM11 .

What sample types have been validated with TRIM11 antibodies?

Most commercially available TRIM11 antibodies have been validated primarily with human samples . Specifically:

• Cell lines: HEK-293, HeLa, and Jurkat cells have shown positive Western blot detection

• Tissues: Human colon cancer tissue has been successfully used for IHC applications

• Some antibodies may cross-react with mouse samples, though this should be experimentally verified for each antibody

When working with other species or sample types, preliminary validation experiments are strongly recommended.

How can researchers effectively use TRIM11 antibodies to investigate its role in cancer progression?

TRIM11 has been identified as an oncogene in non-small cell lung cancer (NSCLC) and other malignancies, with increased expression correlating with poor clinical survival . To investigate TRIM11's role in cancer:

• Expression analysis: Use Western blot and IHC to compare TRIM11 levels between tumor and adjacent normal tissues. Quantify expression differences and correlate with clinical parameters.

• Functional studies: Combine TRIM11 antibodies with knockdown/overexpression approaches. For example, researchers have demonstrated that siRNA-mediated TRIM11 knockdown inhibits cancer cell proliferation, induces apoptosis, and prevents glucose uptake in lung cancer cells .

• Mechanistic investigations: Employ co-immunoprecipitation with TRIM11 antibodies to identify novel binding partners in cancer cells. Research has shown that TRIM11 regulates lung cancer progression via the DUSP6-mediated ERK1/2 signaling pathway .

• Therapeutic potential assessment: Monitor TRIM11 levels after treatment with potential therapeutic agents to determine if TRIM11 downregulation correlates with treatment response.

When designing these experiments, researchers should consider both the catalytic (E3 ligase) and non-catalytic functions of TRIM11, as both may contribute to its oncogenic properties.

What are the critical considerations for optimizing immunohistochemistry protocols with TRIM11 antibodies?

Successful IHC with TRIM11 antibodies requires careful optimization:

• Antigen retrieval: Published protocols recommend using either TE buffer (pH 9.0) or citrate buffer (pH 6.0) for antigen retrieval . Comparative testing of both conditions is advisable for new tissue types.

• Antibody dilution: Start with the recommended range (1:250-1:1000) and optimize based on signal-to-noise ratio .

• Detection system selection: Choose between chromogenic (DAB) or fluorescent detection based on experimental needs. For multiplex staining, consider fluorescent detection to allow co-localization studies with other markers.

• Controls: Always include:

  • Positive control (human colon cancer tissue has been validated)

  • Negative control (primary antibody omission)

  • Blocking peptide control when available

• Counterstaining optimization: Adjust hematoxylin intensity to maintain visibility of TRIM11 staining while providing structural context.

For quantitative analysis, consider digital image analysis using specialized software to measure staining intensity and distribution, particularly when comparing expression across different tissue samples or experimental conditions.

How can researchers address potential non-specific binding when using TRIM11 antibodies?

Non-specific binding can complicate interpretation of results when using TRIM11 antibodies. Advanced troubleshooting approaches include:

• Antibody validation using genetic controls:

  • Use TRIM11 knockout (KO) or knockdown (KD) samples as negative controls

  • Multiple publications have utilized TRIM11 KD/KO approaches that can serve as reference points

• Cross-reactivity assessment:

  • TRIM family proteins share structural similarities that may lead to cross-reactivity

  • Test specificity through Western blot analysis of samples overexpressing different TRIM family members

• Optimization strategies:

  • Increase blocking duration and concentration (5% BSA or 5% normal serum from the same species as the secondary antibody)

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Include negative controls with isotype-matched IgG from the same species as the primary antibody

• Signal validation:

  • Confirm results with a second TRIM11 antibody recognizing a different epitope

  • Verify that the molecular weight of detected bands matches the expected size (53 kDa)

  • For immunofluorescence, co-stain with antibodies against known TRIM11 interacting partners for co-localization assessment

Validation through multiple techniques (e.g., verifying IF results with Western blot) provides stronger evidence for antibody specificity.

What experimental approaches can researchers use to study TRIM11's E3 ubiquitin ligase activity?

Investigating TRIM11's E3 ligase function requires specialized experimental designs:

• In vitro ubiquitination assays:

  • Components: Purified E1, E2 enzyme (typically UbcH5 family), TRIM11 (immunoprecipitated using anti-TRIM11 antibodies), substrate protein, ubiquitin, ATP

  • Detection: Western blot for ubiquitinated substrate using substrate-specific antibodies

  • Controls: Reactions lacking ATP, E1, E2, or using catalytically inactive TRIM11 mutants

• Cellular ubiquitination assays:

  • Co-express tagged ubiquitin, TRIM11, and potential substrate

  • Immunoprecipitate substrate under denaturing conditions

  • Detect ubiquitination by Western blot using anti-ubiquitin or anti-tag antibodies

  • Compare wild-type TRIM11 with RING domain mutants lacking E3 ligase activity

• Proteasome inhibition experiments:

  • Treat cells with proteasome inhibitors (MG132 or bortezomib)

  • Compare substrate levels with/without TRIM11 overexpression or knockdown

  • TRIM11 has been shown to promote degradation of various proteins including PAX6, MED15, and AIM2

• Deubiquitinase interaction studies:

  • TRIM11 interacts with USP14, preventing its association with the proteasome

  • Co-immunoprecipitation with TRIM11 antibodies can identify interactions with deubiquitinases

  • Functional assays can determine how these interactions affect substrate degradation

When publishing these results, researchers should include full gel images and appropriate controls to demonstrate specificity of the observed ubiquitination activity.

What are the best practices for storage and handling of TRIM11 antibodies to maintain optimal activity?

To preserve antibody functionality and extend shelf-life:

• Storage conditions:

  • Store at -20°C in manufacturer-recommended buffer (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

  • Antibodies are generally stable for one year after shipment when stored properly

  • For small volume antibodies (e.g., 20μl), some manufacturers include 0.1% BSA for additional stability

• Handling practices:

  • Avoid repeated freeze-thaw cycles (aliquot upon first thaw if necessary)

  • Centrifuge briefly after thawing to collect all liquid at the bottom of the tube

  • Handle at recommended temperature (usually 4°C) during experiments

  • Return to -20°C promptly after use

• Working dilution preparation:

  • Dilute only the amount needed for immediate use

  • Use high-quality, freshly prepared buffers

  • Add carrier protein (0.1-0.5% BSA) to diluted antibody solutions when working at very low concentrations

• Contamination prevention:

  • Use sterile technique when handling antibodies

  • Avoid introduction of bacteria or fungi which may produce proteases

  • If contamination is suspected, filter through a 0.22μm filter (note this may reduce antibody concentration)

Following these practices will help ensure consistent results across experiments and maximize the usable life of TRIM11 antibodies.

How can researchers troubleshoot weak or absent signals when using TRIM11 antibodies in Western blot?

When facing detection challenges with TRIM11 antibodies in Western blot:

• Sample preparation optimization:

  • Ensure complete cell lysis using appropriate detergents (RIPA buffer is often effective)

  • Include protease inhibitors to prevent degradation

  • Normalize protein loading (25-50μg total protein per lane typically works well)

  • Verify TRIM11 expression in your cell type/tissue (HEK-293, HeLa, and Jurkat cells are known to express detectable levels)

• Protocol adjustments:

  • Try different blocking agents (5% non-fat milk vs. 5% BSA)

  • Optimize primary antibody concentration (try the upper end of the recommended range: 1:1000)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use more sensitive detection systems (enhanced chemiluminescence or fluorescent secondary antibodies)

• Transfer efficiency verification:

  • Check transfer by staining membrane with Ponceau S

  • Ensure transfer conditions are appropriate for a 53 kDa protein

  • Consider using PVDF membrane instead of nitrocellulose for potentially better protein retention

• Technical considerations:

  • Verify antibody functionality with a positive control lysate

  • Test alternative antibody clones that recognize different epitopes

  • Consider the impact of protein modifications or isoforms that might affect epitope accessibility

If TRIM11 levels are expected to be very low, signal amplification systems or more sensitive detection methods may be necessary.

What approaches can researchers use to quantify TRIM11 expression levels across different experimental conditions?

For accurate quantification of TRIM11 expression:

• Western blot densitometry:

  • Capture images within the linear range of detection

  • Use image analysis software (ImageJ, Image Lab, etc.) for densitometry

  • Normalize TRIM11 signal to loading controls (β-actin, GAPDH, or total protein stain)

  • Include a standard curve with known amounts of recombinant TRIM11 for absolute quantification

  • Report data as fold-change relative to control conditions

• Quantitative immunofluorescence:

  • Use consistent image acquisition parameters

  • Measure mean fluorescence intensity in defined cellular compartments

  • Include internal controls in each image

  • Analyze sufficient cell numbers for statistical significance (typically >50 cells per condition)

• Flow cytometry:

  • Optimize fixation and permeabilization for intracellular TRIM11 detection

  • Include fluorescence-minus-one (FMO) controls

  • Report data as median fluorescence intensity (MFI)

  • Consider dual staining with cell type markers for heterogeneous samples

• qRT-PCR for mRNA levels:

  • Design specific primers for TRIM11

  • Use multiple reference genes for normalization

  • Correlate mRNA levels with protein expression to account for post-transcriptional regulation

For all quantification methods, perform at least three biological replicates, conduct appropriate statistical analysis, and consider the dynamic range of the detection method relative to expected expression differences.

How should researchers select the most appropriate TRIM11 antibody for their specific experimental needs?

Strategic antibody selection is critical for experimental success:

• Epitope consideration:

  • For full-length protein detection: Antibodies targeting conserved domains

  • For isoform specificity: Antibodies recognizing unique regions

  • For functional studies: Antibodies targeting the RING domain (for E3 ligase activity) or specific functional motifs

• Application-specific selection:

  • For WB: Antibodies validated with denaturing conditions

  • For IP: Antibodies that recognize native conformations

  • For IHC: Antibodies validated with fixation and antigen retrieval protocols similar to your planned procedure

  • For multiplexing: Consider host species compatibility with other antibodies in your panel

• Validation assessment:

  • Review publication record of the antibody (cited publications)

  • Examine validation data provided by manufacturers

  • Look for validation using genetic controls (KO/KD)

  • Consider antibodies validated in applications and cell/tissue types similar to your experiment

• Technical specifications:

  • Clonality: Polyclonal for higher sensitivity, monoclonal for consistency

  • Host species: Consider compatibility with your experimental system

  • Format: Unconjugated vs. directly labeled

  • Purification method: Affinity-purified antibodies typically offer higher specificity

The search results indicate that rabbit polyclonal antibodies have been extensively validated for TRIM11 detection across multiple applications, making them a good starting point for most research projects .

What are the considerations when investigating TRIM11's role in cancer using tissue microarrays and patient samples?

When examining TRIM11 in cancer contexts:

• Study design considerations:

  • Include sufficient sample sizes for statistical power

  • Match cases and controls for relevant variables (age, gender, stage)

  • Include multiple cancer types to assess tissue specificity (TRIM11 has been implicated in lung cancer)

  • Correlate TRIM11 expression with clinical outcomes and survival data

• Technical optimization for patient tissues:

  • Test multiple antibody dilutions on representative samples before processing entire cohorts

  • Optimize antigen retrieval methods for preserved tissues (FFPE samples may require stronger retrieval conditions)

  • Include on-slide positive and negative controls

  • Consider dual staining with tumor markers for accurate identification of neoplastic cells

• Analysis approaches:

  • Develop consistent scoring systems (H-score, Allred score, or digital quantification)

  • Consider subcellular localization of TRIM11 staining

  • Correlate protein expression with mRNA data when available

  • Integrate with other molecular markers (eg., ERK1/2 pathway components in lung cancer)

• Mechanistic validation:

  • Follow up tissue findings with functional studies in relevant cell lines

  • Test whether TRIM11 knockdown affects cancer-specific phenotypes

  • Investigate downstream pathways (TRIM11 has been shown to regulate the DUSP6-ERK1/2 pathway in lung cancer)

Research has demonstrated that increased expression of TRIM11 correlates with poor clinical survival in cancer patients, highlighting the importance of these investigations .

How can researchers investigate the role of TRIM11 in viral restriction and immune regulation?

TRIM11 has been implicated in antiviral defense and immune regulation . To explore these functions:

• Viral infection studies:

  • Compare viral replication in cells with normal, overexpressed, or knocked-down TRIM11

  • Focus on retroviruses (HIV-1, murine leukemia virus) where TRIM11 has demonstrated effects

  • Assess viral gene expression and virion production

  • Determine whether E3 ligase activity is required for antiviral function using catalytically inactive mutants

• TRIM11-AIM2 inflammasome regulation:

  • TRIM11 inhibits the AIM2 inflammasome by promoting autophagy-dependent degradation of AIM2

  • Monitor inflammasome activation (IL-1β production, ASC speck formation) in relation to TRIM11 levels

  • Track AIM2 localization to autophagosomes using co-localization studies

  • Investigate TRIM11 autoubiquitination upon DNA stimulation

• Mechanistic studies:

  • Examine TRIM11 interaction with SQSTM1/p62 in the context of autophagy induction

  • Investigate how TRIM11 regulates TRIM5 turnover via the proteasome pathway

  • Determine how this affects cross-species restriction of retroviral infection

• Physiological relevance:

  • Compare TRIM11 expression during viral infection or inflammatory stimulation

  • Assess TRIM11 levels in immune cells vs. non-immune cells

  • Investigate potential polymorphisms in TRIM11 that correlate with infection susceptibility

These approaches will help clarify TRIM11's complex roles in innate immunity and antiviral defense mechanisms.

What experimental designs can researchers employ to study the heat shock response regulation by TRIM11?

TRIM11 is upregulated upon heat shock and promotes cell survival . To investigate this stress response function:

• Heat shock induction protocols:

  • Compare acute (42°C for 1-2 hours) vs. chronic (39-40°C for 12-24 hours) heat stress

  • Monitor TRIM11 protein and mRNA levels at different timepoints post-heat shock

  • Include recovery phases to assess persistence of upregulation

  • Compare with other stress conditions (oxidative stress, ER stress) to determine specificity

• Transcriptional regulation analysis:

  • Identify heat shock elements (HSEs) in the TRIM11 promoter region

  • Perform chromatin immunoprecipitation for heat shock factors (HSFs)

  • Use reporter assays to verify functional importance of identified elements

  • Determine if TRIM11 is a direct target of classical heat shock transcription factors

• Functional significance assessment:

  • Compare cell survival after heat shock in TRIM11-normal vs. depleted cells

  • Examine protein aggregation levels as TRIM11 promotes degradation of insoluble proteins

  • Investigate interaction of TRIM11 with heat shock proteins

  • Assess whether TRIM11's proteasome-activating function is enhanced during heat stress

• Mechanism exploration:

  • Compare global ubiquitination profiles before and after heat shock in relation to TRIM11 levels

  • Identify heat-specific TRIM11 substrates

  • Determine if TRIM11's interaction with USP14 and the proteasome is altered during stress conditions

These experiments will help elucidate how TRIM11 contributes to cellular resilience during heat stress and potentially other proteotoxic stress conditions.

How can researchers address cross-reactivity concerns with TRIM11 antibodies?

The TRIM protein family contains over 70 members with similar domain structures, raising cross-reactivity concerns:

• Bioinformatic analysis:

  • Perform sequence alignment of the immunogen region against other TRIM family members

  • Identify potential cross-reactive proteins based on epitope similarity

  • Pay particular attention to closely related TRIMs (TRIM3, TRIM25, TRIM56)

• Experimental verification:

  • Test antibody specificity in TRIM11 knockout/knockdown systems

  • Check reactivity in cells overexpressing different TRIM family members

  • Perform peptide competition assays using the immunizing peptide

• Validation across multiple techniques:

  • Compare antibody performance in techniques with different protein conformations (native vs. denatured)

  • Verify that the molecular weight of detected bands matches the expected 53 kDa

  • Confirm subcellular localization patterns are consistent with known TRIM11 distribution

• Alternative detection strategies:

  • Use epitope-tagged TRIM11 constructs and tag-specific antibodies for validation

  • Consider RNA-based detection methods (RNA-FISH, qRT-PCR) as complementary approaches

  • When possible, verify key findings with multiple TRIM11 antibodies recognizing different epitopes

These approaches will help ensure that observed signals are specific to TRIM11 rather than related TRIM family proteins.

What controls are essential when using TRIM11 antibodies for quantitative or semi-quantitative analyses?

Robust controls are critical for reliable quantitative analyses:

• Specificity controls:

  • TRIM11 knockdown/knockout samples as negative controls

  • TRIM11 overexpression samples as positive controls

  • Isotype control antibodies to assess non-specific binding

  • Secondary antibody-only controls to detect background

• Technical controls:

  • Loading controls for Western blot (β-actin, GAPDH, or total protein stains)

  • Internal reference cells/tissues with known TRIM11 expression levels

  • Standard curves using recombinant TRIM11 for absolute quantification

  • Dilution series to verify detection linearity

• Biological controls:

  • Cell lines with verified TRIM11 expression (HEK-293, HeLa, Jurkat)

  • Tissues with known TRIM11 expression patterns

  • Experimental conditions that alter TRIM11 levels (e.g., heat shock)

  • Multiple biological replicates to account for natural variation

• Data analysis controls:

  • Blinded quantification to prevent bias

  • Multiple independent analysts for critical experiments

  • Statistical tests appropriate for data distribution

  • Inclusion of all replicate data points in addition to means/medians

Implementation of these controls will enhance the reliability and reproducibility of quantitative analyses involving TRIM11 antibodies.

How can researchers validate the specificity of their TRIM11 antibody-based findings?

Comprehensive validation strategies include:

• Genetic approaches:

  • Confirm findings in TRIM11 knockout models (CRISPR/Cas9)

  • Use siRNA/shRNA-mediated knockdown with rescue experiments

  • Employ multiple siRNA sequences targeting different regions of TRIM11

  • Compare results with published TRIM11 KD/KO data

• Multi-antibody validation:

  • Replicate key findings using antibodies recognizing different TRIM11 epitopes

  • Compare monoclonal and polyclonal antibodies when available

  • Verify consistency between commercial sources

• Orthogonal techniques:

  • Complement protein detection with mRNA analysis

  • Use proximity ligation assays to verify protein-protein interactions

  • Employ mass spectrometry for unbiased protein identification

• Functional validation:

  • Assess whether observed phenotypes align with known TRIM11 functions

  • Test domain-specific mutants to connect findings to specific TRIM11 activities

  • Investigate whether manipulation of TRIM11 levels affects expected downstream targets

• Publication standards:

  • Report all antibody validation data

  • Include detailed methods for reproducibility

  • Disclose limitations and potential cross-reactivity issues

These validation approaches will strengthen the reliability of TRIM11 antibody-based discoveries and enhance their scientific impact.