TRIM71 Antibody, HRP conjugated

What is TRIM71 and why is it important in research?

TRIM71 (Tripartite Motif Containing 71, E3 Ubiquitin Protein Ligase) is a protein that plays crucial roles in embryonic stem cell (ESC) maintenance and developmental processes. It functions primarily as an E3 ubiquitin-protein ligase that cooperates with the microRNA machinery to promote embryonic stem cell proliferation and maintenance . Research has shown that TRIM71 enhances FGF/ERK signaling by stabilizing SHCBP1, which is important for understanding stem cell biology and developmental processes . TRIM71 deficiency has also been associated with human male infertility and germ cell loss, highlighting its importance in reproductive biology .

What applications are HRP-conjugated TRIM71 antibodies suitable for?

HRP-conjugated TRIM71 antibodies are particularly suitable for several immunodetection methods:

• Western Blotting (WB): Provides high sensitivity detection of TRIM71 protein expression levels

• ELISA: Allows quantitative measurement of TRIM71 in biological samples

• Immunohistochemistry (IHC): Enables visualization of TRIM71 localization in tissue sections

The HRP conjugation eliminates the need for secondary antibody incubation, reducing background and simplifying workflows . When using these antibodies, researchers should consider cross-reactivity profiles, as some TRIM71 antibodies show reactivity across multiple species including human, mouse, rat, and other mammals .

What domains and regions of TRIM71 are typically recognized by antibodies?

TRIM71 antibodies are typically designed to recognize specific domains within the protein:

• C-Terminal region antibodies: These target the C-terminal portion which includes the NHL domain, critical for RNA binding and protein-protein interactions

• Mid-region antibodies: Target amino acids 125-209 or 390-402, depending on the antibody

• N-Terminal region antibodies: Recognize the TRIM motif which consists of a RING domain, two B-boxes, and a coiled-coil (CC) domain

The choice of domain-specific antibody is important as TRIM71's functions vary by domain. For instance, the NHL domain is required for binding to long noncoding RNA Trincr1 and regulating FGF/ERK signaling, while the RING domain is involved in ubiquitin ligase activity .

How should I optimize Western blot conditions for HRP-conjugated TRIM71 antibodies?

Optimizing Western blot conditions for HRP-conjugated TRIM71 antibodies requires careful attention to several parameters:

• Protein Extraction and Loading:

  • Use RIPA buffer supplemented with protease inhibitors to efficiently extract TRIM71

  • Load 20-40 μg of total protein per lane for cell lysates

  • Include positive controls (e.g., embryonic stem cell lysates) where TRIM71 is known to be expressed

• Electrophoresis and Transfer:

  • Use 8-10% SDS-PAGE gels as TRIM71 has a molecular weight of approximately 110 kDa

  • Transfer at 100V for 60-90 minutes to PVDF membrane (preferred over nitrocellulose for this protein)

• Blocking and Antibody Incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Dilute HRP-conjugated TRIM71 antibody at 1:1000 to 1:5000 depending on the specific product

  • Incubate overnight at 4°C for optimal results

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate compatible with HRP

  • Start with short exposure times (30 seconds) and increase as needed

This methodology has been validated in studies examining TRIM71's role in ERK signaling pathways .

What controls are essential when using TRIM71 antibodies in experimental setups?

When designing experiments with TRIM71 antibodies, several controls are essential to ensure valid and interpretable results:

• Positive Controls:

  • Embryonic stem cell (ESC) lysates: ESCs show high endogenous expression of TRIM71

  • Recombinant TRIM71 protein: Can serve as a standard for antibody validation

  • HEK293 cells overexpressing FLAG-tagged TRIM71: Used in co-IP experiments

• Negative Controls:

  • TRIM71 knockdown samples: Using siRNA, shRNA, or CRISPR/Cas9 systems to validate antibody specificity

  • Tissues known to not express TRIM71: Adult differentiated tissues generally express lower levels

  • Isotype control antibodies: Should show no specific binding

• Technical Controls:

  • Loading controls (e.g., β-actin, GAPDH) for Western blotting

  • No primary antibody control to assess non-specific binding of secondary reagents

  • Peptide competition assay: Pre-incubation with the immunizing peptide should abolish specific signal

Implementing these controls is critical for distinguishing between genuine TRIM71 detection and non-specific signals.

How can I validate TRIM71 antibody specificity for my particular research model?

Validating TRIM71 antibody specificity requires a multi-faceted approach:

• Genetic Validation:

  • Generate TRIM71 knockdown/knockout models using:

    • siRNA/shRNA approaches: Multiple studies have used this approach to validate TRIM71 function in ESCs

    • CRISPR/Cas9-mediated deletion: CRISPRi strategy with two gRNAs has been effective for TRIM71 knockdown

  • Compare antibody signal between wild-type and TRIM71-depleted samples

• Molecular Weight Verification:

  • TRIM71 should be detected at approximately 110 kDa on Western blots

  • Verify that truncated forms in deletion studies show appropriate size changes

• Domain-Specific Validation:

  • Use cells expressing TRIM71 truncation mutants lacking specific domains (RING, NHL)

  • Confirm that antibodies targeting specific domains fail to detect corresponding deletion mutants

• Cross-Species Reactivity Testing:

  • If working with non-human models, verify antibody reactivity in your species

  • Predicted reactivity may vary: Mouse (93%), Human (100%), Rat (100%)

Proper validation ensures experimental observations genuinely reflect TRIM71 biology rather than antibody artifacts.

How can I use TRIM71 antibodies to investigate its interaction with non-coding RNAs?

Investigating TRIM71 interactions with non-coding RNAs requires specialized methodologies:

• RNA Immunoprecipitation (RIP) using TRIM71 antibodies:

  • Cross-link RNA-protein complexes using formaldehyde or UV

  • Immunoprecipitate TRIM71 using validated antibodies (non-HRP conjugated)

  • Extract bound RNAs and analyze by qPCR or sequencing

  • This approach has successfully identified Trincr1 (TRIM71 interacting long noncoding RNA 1) with ~70-fold enrichment in IP versus input

• RNA Pull-down Assays:

  • Synthesize biotinylated RNA of interest by in vitro transcription

  • Incubate with cell lysates and capture using streptavidin beads

  • Detect TRIM71 in pulled-down complexes using Western blotting

  • Control RNAs (antisense or unrelated RNAs like Malat1) should be included as specificity controls

• Mapping Interaction Domains:

  • Use TRIM71 truncation constructs to identify RNA-binding domains

  • Studies have shown that the NHL domain of TRIM71 is required for binding to Trincr1

  • RNA truncation experiments can identify the minimal RNA sequence required for binding

This methodology has revealed that TRIM71 binds to the 5' half of Trincr1, which represses FGF/ERK signaling in ESCs .

What approaches can resolve contradictory data regarding TRIM71 function in different cell types?

Resolving contradictory data regarding TRIM71 function across different cellular contexts requires systematic approaches:

• Cell Type-Specific Analysis:

  • Use HRP-conjugated TRIM71 antibodies for quantitative comparison of expression levels

  • Compare subcellular localization across cell types using immunofluorescence

  • Embryonic stem cells show high TRIM71 expression with roles in promoting FGF/ERK signaling

  • In contrast, neural progenitor cells and hepatocellular carcinoma cells may show different regulatory patterns

• Interactome Analysis by Cell Type:

  • Perform immunoprecipitation with TRIM71 antibodies followed by mass spectrometry

  • Compare TRIM71-interacting proteins between different cell types

  • Analyze post-translational modifications that might affect function

• Target mRNA Comparison:

  • TRIM71 regulates different mRNAs in different contexts:

    • Stabilizes SHCBP1 in ESCs

    • Regulates Blimp1 in germ cells

  • Perform RIP-Seq across cell types to identify differential RNA targets

• Mechanistic Reconciliation:

  • Test if contradictions stem from:

    • Different TRIM71 isoforms expressed in different cells

    • Varying cofactors that modify TRIM71 activity

    • Context-dependent post-translational modifications

This comprehensive approach has helped clarify that TRIM71 can both enhance and repress signaling pathways depending on cellular context and interacting partners .

How can TRIM71 antibodies help elucidate the protein's role in developmental disorders and disease models?

TRIM71 antibodies are powerful tools for investigating the protein's involvement in developmental disorders and disease models:

• Developmental Phenotype Analysis:

  • Use HRP-conjugated TRIM71 antibodies for immunohistochemistry on developmental tissue sections

  • Compare TRIM71 expression patterns between normal and pathological samples

  • TRIM71 deficiency has been linked to male infertility through disrupted germ cell development

  • Track TRIM71 expression during critical developmental windows

• Signaling Pathway Dysregulation:

  • Analyze FGF/ERK signaling components in TRIM71-deficient models

  • Measure phosphorylated ERK levels by Western blotting

  • TRIM71 promotes FGF/ERK signaling in ESCs, and its dysregulation may contribute to developmental defects

• Disease Model Applications:

  • Cancer biology: Examine TRIM71 levels in tumor samples, as altered expression may affect proliferation pathways

  • Reproductive disorders: Analyze TRIM71 expression in infertility cases

  • Developmental disorders: Investigate TRIM71 in congenital abnormalities

• Therapeutic Target Validation:

  • Use TRIM71 antibodies to validate target engagement in drug discovery

  • Monitor TRIM71 levels and localization following experimental interventions

  • Assess pathway normalization in rescue experiments

These approaches help establish mechanistic links between TRIM71 dysfunction and disease phenotypes, potentially identifying new therapeutic targets.

What are common pitfalls when using HRP-conjugated TRIM71 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with HRP-conjugated TRIM71 antibodies:

• High Background Issues:

  • Cause: Insufficient blocking or excessive antibody concentration

  • Solution: Optimize blocking (try 5% BSA instead of milk for phospho-specific applications), increase wash time/frequency, and titrate antibody from 1:1000 to 1:5000 to determine optimal concentration

• Weak or Absent Signal:

  • Cause: Low TRIM71 expression, protein degradation, or inefficient extraction

  • Solution: Use positive control samples (ESCs), include protease inhibitors during extraction, and consider enrichment through immunoprecipitation before detection

• Multiple Bands on Western Blot:

  • Cause: Degradation products, cross-reactivity, or post-translational modifications

  • Solution: Verify with knockout/knockdown controls, try different lysis buffers to reduce degradation, and consider using domain-specific antibodies to distinguish isoforms

• Batch-to-Batch Variability:

  • Cause: Differences in production affecting specificity or sensitivity

  • Solution: Validate each new lot against previous lots using standardized positive controls and maintain consistent detection parameters

• Non-reproducible Co-IP Results:

  • Cause: Transient or weak interactions disrupted during washing

  • Solution: Use gentler wash conditions, chemical crosslinking, or optimize buffer compositions based on known TRIM71 interactions like those with Trincr1

These troubleshooting approaches have been validated in studies examining TRIM71's role in stem cell biology and developmental processes.

How can I optimize RNA-protein interaction studies using TRIM71 antibodies?

Optimizing RNA-protein interaction studies with TRIM71 antibodies requires attention to several key parameters:

• RNA Immunoprecipitation (RIP) Protocol Optimization:

  • Cross-linking: UV at 254 nm provides direct RNA-protein crosslinks, while formaldehyde preserves indirect interactions

  • Lysis conditions: Use NP-40 based buffers with RNase inhibitors to maintain RNA integrity

  • Antibody selection: Non-conjugated antibodies targeting the NHL domain are ideal, as this domain mediates RNA interactions

  • Washing stringency: Balance between removing non-specific interactions without disrupting specific binding

• Controls for RNA-Binding Specificity:

  • Input control: Typically 5-10% of lysate before immunoprecipitation

  • IgG control: Non-specific antibody of same isotype

  • TRIM71 domain mutants: NHL domain mutants should show reduced RNA binding

  • RNase treatment control: Confirms RNA-dependency of interactions

• Sequential Immunoprecipitation for Complex Analysis:

  • First IP with TRIM71 antibody

  • Elution under native conditions

  • Second IP with antibodies against suspected co-factors

  • This approach can identify RNA-dependent protein complexes involving TRIM71

• Quantification and Analysis:

  • qRT-PCR for known targets (e.g., Trincr1)

  • RNA-seq for unbiased discovery of all bound RNAs

  • Bioinformatic analysis to identify binding motifs, such as the hairpin structures recognized by TRIM71

These methodologies have successfully identified Trincr1 as a TRIM71-interacting lncRNA that regulates FGF/ERK signaling in ESCs .

What strategies can improve detection of low-abundance TRIM71 in differentiated cell types?

Detecting low-abundance TRIM71 in differentiated cells presents challenges that can be overcome with specialized approaches:

• Signal Amplification Techniques:

  • Tyramide Signal Amplification (TSA): Enhances sensitivity of HRP-conjugated antibodies by depositing multiple tyramide molecules

  • Poly-HRP conjugated secondary antibodies: Provide greater signal than standard HRP conjugates

  • Biotin-streptavidin systems: Can amplify signal when using biotinylated primary antibodies

• Sample Enrichment Methods:

  • Subcellular fractionation: Concentrate samples based on TRIM71's known cytoplasmic localization

  • Immunoprecipitation followed by Western blotting: Enriches TRIM71 from larger sample volumes

  • Targeted proteomics approaches: Selected Reaction Monitoring (SRM) for specific TRIM71 peptides

• Enhanced Detection Systems:

  • Highly sensitive ECL substrates (e.g., femto-level detection reagents)

  • Digital immunoassay platforms with single-molecule sensitivity

  • Fluorescently-tagged antibodies with confocal microscopy for localization studies

• Optimization for Specific Cell Types:

  • Adjust fixation protocols for immunohistochemistry (different cells may require different fixatives)

  • Optimize antigen retrieval methods for tissue sections

  • Consider cell-specific lysis buffers that effectively extract TRIM71 while minimizing background

These approaches have proven effective in detecting TRIM71 across various cell types including differentiated cells where expression levels are significantly lower than in embryonic stem cells .

How might TRIM71 antibodies contribute to understanding developmental timing regulation?

TRIM71 antibodies can significantly advance our understanding of developmental timing regulation through several research approaches:

• Temporal Expression Profiling:

  • Use HRP-conjugated TRIM71 antibodies to track expression changes during developmental transitions

  • Create precise temporal maps of TRIM71 expression across embryonic stages

  • Correlate expression patterns with key developmental milestones and cell fate decisions

• Interaction Dynamics Investigation:

  • Apply TRIM71 antibodies in time-course immunoprecipitation experiments

  • Identify stage-specific protein interactions that may regulate developmental timing

  • Studies have shown TRIM71 interacts with different partners at different developmental stages

• Post-translational Modification Analysis:

  • Use modification-specific antibodies alongside TRIM71 antibodies

  • Determine how phosphorylation, ubiquitination, or other modifications affect TRIM71 function

  • Track modifications that might serve as molecular switches during development

• In vivo Developmental Studies:

  • Apply TRIM71 antibodies in immunohistochemistry of developmental tissues

  • Track spatial-temporal expression patterns related to organ formation

  • TRIM71 deficiency causes germ cell loss, suggesting critical timing roles in reproductive development

These approaches would build upon findings that TRIM71 regulates FGF/ERK signaling in ESCs and impacts developmental processes through its interaction with non-coding RNAs like Trincr1 .

What emerging technologies might enhance TRIM71 antibody applications in research?

Several emerging technologies promise to expand the utility of TRIM71 antibodies in research:

• Proximity Labeling with TRIM71 Antibodies:

  • BioID or APEX2 systems coupled with TRIM71 recognition

  • Map the spatial proteome surrounding TRIM71 in living cells

  • Identify transient or weak interactions missed by traditional co-IP approaches

• Single-Cell Analysis Applications:

  • Single-cell Western blotting using TRIM71 antibodies

  • CyTOF (mass cytometry) adaptation for deep phenotyping with TRIM71 as a marker

  • Spatial transcriptomics combined with TRIM71 protein detection to correlate protein levels with transcriptional states

• Live-Cell Imaging Innovations:

  • Nanobody or scFv derivatives of TRIM71 antibodies for intracellular tracking

  • FRET-based biosensors to detect TRIM71 interactions with targets in real-time

  • Optogenetic tools to manipulate TRIM71 function while monitoring effects

• Advanced Structural Studies:

  • Cryo-EM studies of TRIM71 complexes using antibody fragments for stabilization

  • Hydrogen-deuterium exchange mass spectrometry with TRIM71 antibodies to probe conformational changes

  • In-cell NMR approaches to study TRIM71 structural dynamics in native environments

These technologies could significantly enhance our understanding of how TRIM71 functions in its native cellular context, particularly in relation to its interactions with RNAs like Trincr1 and its role in developmental processes .

How can TRIM71 antibodies facilitate cross-disciplinary research between stem cell biology and developmental disorders?

TRIM71 antibodies can serve as powerful tools bridging stem cell biology and developmental disorder research:

• Translational Research Applications:

  • Compare TRIM71 expression and localization between patient-derived and healthy samples

  • Use HRP-conjugated antibodies for high-throughput screening of clinical specimens

  • TRIM71 deficiency has been associated with human male infertility, providing a direct clinical connection

• Disease Modeling with iPSCs:

  • Track TRIM71 expression during reprogramming and differentiation

  • Compare TRIM71-mediated pathways between healthy and patient-derived iPSCs

  • Use TRIM71 antibodies to validate disease models representing developmental disorders

• Therapeutic Development Platforms:

  • Screen drug candidates for normalization of TRIM71 expression or function

  • Monitor pathway restoration using TRIM71 antibodies in treated samples

  • Validate gene therapy approaches targeting TRIM71 or its regulatory networks

• Genomics-Proteomics Integration:

  • Correlate genetic variants in TRIM71 with protein expression patterns

  • Investigate how mutations affect TRIM71 interaction with binding partners like Trincr1

  • Examine epigenetic regulation of TRIM71 in development and disease

This cross-disciplinary approach connects fundamental stem cell research with clinical applications, potentially leading to new diagnostic tools and therapeutic strategies for developmental disorders associated with TRIM71 dysfunction .