TRIM71 Antibody, FITC conjugated

What is TRIM71 and why is it a significant research target?

TRIM71 (Tripartite Motif Containing 71) is an E3 ubiquitin-protein ligase that plays crucial roles in embryonic stem cell proliferation and maintenance. It functions cooperatively with the microRNA machinery, binding to miRNAs and associating with AGO2 to participate in post-transcriptional repression of transcripts such as CDKN1A . TRIM71 facilitates the G1-S transition to promote rapid embryonic stem cell self-renewal and is required to maintain proliferation while preventing premature differentiation of neural progenitor cells during early neural development. Additionally, it serves as a specific regulator of miRNA biogenesis, binding to miRNA MIR29A hairpin and post-transcriptionally modulating MIR29A levels, which indirectly regulates TET proteins expression .

Research interest in TRIM71 has grown significantly due to its involvement in developmental processes and its potential role in carcinogenesis, particularly in hepatocellular carcinoma, making TRIM71 antibodies valuable tools for investigating these biological mechanisms .

What are the key specifications of commercially available TRIM71 FITC-conjugated antibodies?

TRIM71 FITC-conjugated antibodies are typically polyclonal antibodies developed in rabbit hosts that specifically target human TRIM71 protein. These antibodies have excitation/emission wavelengths of 499/515 nm and are compatible with the 488 nm laser line commonly used in flow cytometry and fluorescence microscopy applications .

The immunogen for FITC-conjugated TRIM71 antibodies often consists of a recombinant human E3 ubiquitin-protein ligase TRIM71 protein fragment (specifically amino acids 125-209) . These antibodies are generally purified using Protein G affinity chromatography, achieving purity levels greater than 95%. They are typically supplied in a buffer containing 0.01 M PBS (pH 7.4), 0.03% Proclin-300, and 50% glycerol to maintain stability during storage .

How does TRIM71's function relate to its potential as a biomarker in research?

TRIM71's multifaceted roles in cellular processes position it as a valuable biomarker in several research contexts. TRIM71 regulates let-7 expression and activity through two independent mechanisms: enhancing pre-let-7 degradation through direct interaction with LIN28 and TUT4 (thereby inhibiting let-7 maturation), and repressing the activity of mature let-7 via RNA-dependent interaction with the RNA-induced silencing complex (RISC) effector protein AGO2 .

These regulatory functions make TRIM71 particularly relevant as a biomarker in stem cell research, developmental biology, and cancer studies. In hepatocellular carcinoma, TRIM71 appears to drive carcinogenesis, suggesting its potential as a diagnostic or prognostic biomarker . Additionally, recent research has revealed an association between TRIM71 deficiency and male infertility, highlighting its potential as a biomarker in reproductive biology research . The diverse functions of TRIM71 in controlling cell proliferation, particularly in germ cell tumor-derived cells, further emphasize its value as a research biomarker for understanding both developmental processes and malignancy development .

What are the recommended protocols for using FITC-conjugated TRIM71 antibodies in flow cytometry experiments?

When utilizing FITC-conjugated TRIM71 antibodies for flow cytometry experiments, researchers should adhere to several key methodological considerations. First, sample preparation is crucial - cells should be fixed with 4% paraformaldehyde for 10-15 minutes at room temperature, followed by permeabilization using 0.1% Triton X-100 for intracellular staining, as TRIM71 is predominantly an intracellular protein .

For the staining procedure, begin by washing cells twice with PBS containing 1% BSA. Block non-specific binding by incubating cells with 10% normal serum from the same species as the secondary antibody (if using) in PBS for 30 minutes. The FITC-conjugated TRIM71 antibody should be diluted to an appropriate concentration (typically 1-10 μg/ml, though optimal dilutions should be determined empirically by each laboratory) . Incubate cells with the diluted antibody for 30-60 minutes at room temperature or 4°C in the dark to preserve the fluorescence of the FITC conjugate.

After incubation, wash cells thoroughly (3-4 times) with PBS containing 1% BSA to remove unbound antibody. For flow cytometric analysis, use a 488 nm laser for excitation and detect emission at approximately 515 nm . Include appropriate negative controls (isotype-matched FITC-conjugated IgG) and positive controls to ensure specificity and accuracy of the results. When analyzing data, consider using compensation controls if performing multi-color flow cytometry to correct for spectral overlap.

How can researchers effectively validate the specificity of TRIM71 antibodies in their experimental systems?

Validating antibody specificity is critical for generating reliable data. For TRIM71 antibodies, a multi-faceted validation approach is recommended. First, perform Western blotting analysis using cell lysates from cells known to express TRIM71 (such as embryonic stem cells or specific cancer cell lines) alongside negative control cells with low or no TRIM71 expression . The antibody should detect a single band of approximately 94 kDa, corresponding to the molecular weight of TRIM71.

Second, implement genetic knockdown or knockout validation by utilizing siRNA-mediated knockdown of TRIM71 (as demonstrated in TCam-2 cells) or CRISPR/Cas9-mediated knockout, followed by immunoblotting or immunofluorescence to confirm reduced or absent signal compared to wild-type cells. This approach provides strong evidence for antibody specificity .

Third, consider peptide competition assays wherein the TRIM71 antibody is pre-incubated with an excess of the immunizing peptide prior to use in Western blotting or immunostaining. If the antibody is specific, pre-incubation with the peptide should significantly reduce or eliminate the signal.

Fourth, utilize orthogonal validation methods such as RNA-protein correlation, where TRIM71 protein levels detected by the antibody are compared with TRIM71 mRNA levels measured by RT-qPCR across multiple samples . A positive correlation supports antibody specificity. Finally, consider cross-validation using multiple antibodies targeting different epitopes of TRIM71 to confirm consistent detection patterns .

What are the critical considerations for co-localization studies using FITC-conjugated TRIM71 antibodies?

When conducting co-localization studies with FITC-conjugated TRIM71 antibodies, several methodological considerations are essential for obtaining reliable results. First, spectral compatibility must be carefully planned. Since FITC has an emission peak around 515 nm , companion fluorophores should have minimal spectral overlap—consider fluorophores such as Cy3, Texas Red, or Alexa Fluor 594 for co-staining experiments to avoid bleed-through and false co-localization signals.

Sample preparation and fixation methods significantly impact co-localization accuracy. For TRIM71 studies, 4% paraformaldehyde fixation is generally recommended, but researchers should be aware that different fixation protocols may affect epitope accessibility and protein localization. Therefore, validation with multiple fixation methods might be necessary, especially when studying potential interactions between TRIM71 and its binding partners like LIN28, TUT4, or AGO2 .

When imaging, confocal microscopy with appropriate negative controls is strongly recommended over widefield fluorescence for co-localization studies. These controls should include single-stained samples to set acquisition parameters and secondary-antibody-only controls to assess background fluorescence. Additionally, given TRIM71's interaction with RNA and RNA-binding proteins , RNase treatment controls may be valuable to distinguish between direct protein-protein interactions and RNA-mediated associations.

For quantitative co-localization analysis, use established coefficients such as Pearson's correlation coefficient, Mander's overlap coefficient, or intensity correlation analysis. When interpreting results, consider that TRIM71 has been shown to interact with multiple protein complexes in different cellular compartments, including the miRNA processing machinery and the lncRNA Trincr1 , which may result in distinct co-localization patterns depending on cellular context and physiological state.

How should researchers interpret variations in TRIM71 expression patterns across different cell types?

When interpreting variations in TRIM71 expression across different cell types, researchers should consider several biological contexts. TRIM71 exhibits tissue-specific and developmental stage-specific expression patterns. It is highly expressed in embryonic stem cells and plays critical roles in maintaining their self-renewal capacity . The expression typically decreases upon differentiation, making TRIM71 a potential marker for stemness.

In cancer research, elevated TRIM71 expression has been observed in hepatocellular carcinoma and certain germ cell tumors . For instance, analysis using the DepMap database has revealed significant correlations between TRIM71 expression and cell proliferation in liver cancer cells . When comparing expression data across cell types, researchers should normalize to appropriate reference genes and consider both the relative abundance and the functional consequences of differential expression.

Context-dependent interactions significantly impact TRIM71 function. TRIM71's interaction with the let-7 repressor complex (LIN28/TUT4) affects miRNA processing , while its binding to the lncRNA Trincr1 influences FGF/ERK signaling . Therefore, researchers should examine not only TRIM71 expression but also the expression of these interaction partners when interpreting experimental results. Cell-type-specific differences in these partner molecules can lead to different functional outcomes despite similar TRIM71 expression levels.

When analyzing immunofluorescence data with FITC-conjugated TRIM71 antibodies, subcellular localization patterns should be carefully assessed. TRIM71 has been observed in both cytoplasmic and nuclear compartments, with its distribution potentially reflecting different functional states. Quantitative analysis of signal intensity and co-localization with known markers (like P-bodies for post-transcriptional regulation functions) provides valuable information beyond simple expression levels .

What are common technical challenges when using FITC-conjugated antibodies and how can they be addressed?

Working with FITC-conjugated TRIM71 antibodies presents several technical challenges that researchers should anticipate and address methodically. Photobleaching is a primary concern as FITC is relatively susceptible to photobleaching compared to other fluorophores. To mitigate this issue, minimize exposure to light during all experimental procedures by working in reduced lighting conditions and covering samples with aluminum foil . Additionally, incorporate anti-fade mounting media containing agents like p-phenylenediamine or commercial products like ProLong Gold when preparing slides for microscopy. When acquiring images, use the lowest laser power and shortest exposure time that still yields acceptable signal-to-noise ratios.

Autofluorescence in the green channel can significantly confound FITC signal interpretation, particularly in tissues with high flavin or NAD(P)H content. To address this, implement proper controls including unstained samples to establish baseline autofluorescence levels. Consider using spectral unmixing algorithms during image analysis to separate true FITC signal from autofluorescence. For tissues with particularly high autofluorescence, treatment with sodium borohydride (NaBH₄) or photobleaching of the sample prior to antibody application may reduce background.

pH sensitivity represents another challenge as FITC fluorescence intensity decreases significantly at lower pH values, potentially giving misleading results in acidic cellular compartments. Maintain consistent pH (ideally around 7.4) in all buffers used during the experiment . For experiments involving lysosomes or other acidic organelles, consider using alternative fluorophores like Alexa Fluor 488, which exhibits greater pH stability.

Cross-reactivity concerns can arise even with validated antibodies. To address potential non-specific binding, implement rigorous blocking procedures using 5-10% normal serum from the same species as the secondary antibody (if using a detection system) or 3-5% BSA in PBS. Additionally, include appropriate negative controls such as isotype-matched FITC-conjugated IgG antibodies at the same concentration as the TRIM71 antibody .

How can researchers reconcile contradictory findings regarding TRIM71's role in different experimental models?

Reconciling contradictory findings regarding TRIM71's functions requires careful consideration of several factors. First, context-dependent molecular interactions significantly influence TRIM71 activity. For instance, TRIM71's effect on let-7 miRNA processing appears contradictory in different studies: while some research suggests TRIM71 enhances pre-let-7 degradation through interaction with LIN28 and TUT4 , other work has proposed that TRIM71 can mediate ubiquitination and proteasomal degradation of LIN28B, thereby inducing let-7 up-regulation. These seemingly contradictory mechanisms likely reflect cell-type-specific interactions and experimental conditions.

Differential domain functionality also contributes to contradictory findings. TRIM71 contains multiple functional domains, including the RING domain with E3 ubiquitin ligase activity and the NHL domain involved in RNA binding and protein interactions. Research with ΔRING or ΔNHL6 mutants in NCCIT cells demonstrated that these domains have different effects on proliferation, with the ΔNHL6 mutant showing stronger inhibition of proliferation (25.2% reduction) compared to the ΔRING mutant (11.2% reduction) . This suggests that the NHL domain, likely through regulation of CDKN1A, plays a more significant role in proliferation control than the RING domain in this context.

To reconcile contradictory findings, researchers should implement comprehensive experimental designs that examine TRIM71 function across multiple model systems, using both gain-of-function and loss-of-function approaches. Additionally, direct comparative studies between different cell types or developmental stages can help elucidate the context-dependent nature of TRIM71 activity. Single-cell analyses may be particularly valuable for understanding cell-to-cell variability in TRIM71 function, which could explain seemingly contradictory population-level observations .

How can TRIM71 antibodies be utilized to investigate its role in miRNA processing and activity?

TRIM71 antibodies, including FITC-conjugated variants, offer powerful tools for investigating the protein's complex role in miRNA processing and activity. RNA immunoprecipitation (RIP) represents a foundational technique where TRIM71 antibodies can be used to isolate TRIM71-RNA complexes followed by sequencing (RIP-Seq) or qPCR (RIP-qPCR) to identify associated miRNAs and precursors. This approach has successfully identified TRIM71-bound transcripts, including the crucial let-7 miRNA family and other regulatory RNAs .

For studying TRIM71's impact on miRNA processing, researchers can employ co-immunoprecipitation (co-IP) experiments using TRIM71 antibodies to pull down protein complexes, followed by immunoblotting for known miRNA biogenesis factors such as LIN28, TUT4, and AGO2. This approach has revealed that TRIM71 interacts with both LIN28A and LIN28B, key regulators of let-7 processing . Combining TRIM71 antibody-mediated IP with mass spectrometry analysis can further identify novel protein interactions within the miRNA processing machinery.

Advanced microscopy techniques utilizing FITC-conjugated TRIM71 antibodies, such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy, enable high-resolution visualization of TRIM71's co-localization with processing bodies (P-bodies) or stress granules, where post-transcriptional regulation often occurs. Live-cell imaging approaches using anti-TRIM71 antibody fragments can monitor the dynamics of TRIM71-containing complexes during miRNA processing.

For functional studies, researchers can combine TRIM71 antibody-mediated detection with luciferase reporter assays containing let-7 target sequences. This allows quantification of let-7 activity in response to TRIM71 manipulation, providing insights into how TRIM71 modulates miRNA function . Additionally, proximity ligation assays (PLA) using TRIM71 antibodies paired with antibodies against AGO2 or other RISC components can visualize and quantify direct interactions in situ, offering spatial information about where in the cell these regulatory complexes form.

What methodological approaches can reveal TRIM71's mechanistic role in stem cell maintenance and differentiation?

Investigating TRIM71's role in stem cell biology requires sophisticated methodological approaches leveraging TRIM71 antibodies. Chromatin immunoprecipitation sequencing (ChIP-seq) combined with TRIM71 antibodies can identify genomic regions where TRIM71-containing complexes associate with chromatin, potentially revealing direct or indirect transcriptional regulatory mechanisms affecting stemness genes. This approach is particularly relevant given TRIM71's known roles in embryonic stem cell proliferation and maintenance .

Time-course immunofluorescence studies using FITC-conjugated TRIM71 antibodies during directed differentiation protocols provide dynamic information about TRIM71 expression and localization as cells transition from pluripotent to differentiated states. Quantitative image analysis of nuclear versus cytoplasmic TRIM71 signal can reveal translocation events that might signify changing functional roles during differentiation.

For mechanistic studies, CRISPR/Cas9-mediated genome editing to create TRIM71 domain mutants, followed by rescue experiments with wild-type TRIM71, allows precise dissection of domain-specific functions in stem cell maintenance. Subsequent immunoprecipitation using TRIM71 antibodies can identify differential protein interactions of these mutants, connecting structural features to functional roles . Similarly, TRIM71 antibodies can be employed in RNA-protein interaction mapping techniques like CLIP-seq (crosslinking immunoprecipitation followed by sequencing) to identify the RNA targets through which TRIM71 exerts its regulatory effects on stem cell gene expression programs.

Integration of TRIM71 antibody-based detection with single-cell transcriptomics and proteomics creates powerful platforms for understanding heterogeneity in TRIM71's function across subpopulations of stem cells. This approach is particularly valuable for identifying the earliest molecular changes associated with commitment to differentiation and how TRIM71 may regulate these decision points. Additionally, in vitro differentiation assays combined with quantitative immunofluorescence using FITC-conjugated TRIM71 antibodies can reveal how TRIM71 levels correlate with differentiation efficiency into specific lineages, providing insights into its role in cell fate determination .

How can TRIM71 antibodies contribute to understanding its emerging role in cancer biology?

TRIM71 antibodies provide essential tools for exploring the protein's emerging functions in cancer biology. Tissue microarray (TMA) analysis using TRIM71 antibodies enables high-throughput evaluation of TRIM71 expression across multiple tumor types and stages, facilitating correlation with clinical parameters. This approach has proven valuable for hepatocellular carcinoma research, where TRIM71 appears to drive carcinogenesis . Quantitative immunohistochemistry with careful scoring systems allows researchers to determine whether TRIM71 expression levels correlate with tumor grade, stage, or patient outcomes.

For functional studies, researchers can employ TRIM71 antibodies in combination with proliferation assays to assess how TRIM71 manipulation affects cancer cell growth. This methodology has revealed significant proliferation defects in TRIM71-deficient cells, as demonstrated in TCam-2 and NCCIT cell lines, suggesting TRIM71's potential role in promoting the malignancy of germ cell tumors . Cell cycle analysis using flow cytometry with FITC-conjugated TRIM71 antibodies alongside DNA content markers can further elucidate how TRIM71 influences cell cycle progression in cancer cells.

Co-immunoprecipitation experiments using TRIM71 antibodies followed by mass spectrometry or immunoblotting for known oncogenic signaling molecules can identify cancer-specific protein interactions. This approach is particularly relevant given TRIM71's known interactions with critical regulatory factors like AGO2 and various RNA species . Similarly, RIP-seq using TRIM71 antibodies in cancer cell models can identify cancer-specific RNA targets that might contribute to malignant phenotypes.

Xenograft models combined with ex vivo analysis using TRIM71 antibodies enable investigation of TRIM71's role in tumor formation, growth, and metastasis in vivo. Immunofluorescence analysis of tumor sections can reveal spatial heterogeneity in TRIM71 expression and its correlation with proliferative markers, areas of invasion, or cancer stem cell markers. Additionally, pharmacological studies using compounds that modulate pathways upstream or downstream of TRIM71, combined with TRIM71 antibody-based detection methods, can help position TRIM71 within oncogenic signaling networks and potentially identify therapeutic vulnerabilities in TRIM71-expressing tumors .

What emerging techniques might enhance the utility of TRIM71 antibodies in research?

Several cutting-edge methodological approaches are poised to expand the research applications of TRIM71 antibodies. Proximity-dependent biotinylation methods, such as BioID or TurboID fused to TRIM71, combined with antibody-based validation, offer powerful approaches for mapping the TRIM71 protein interactome with spatial and temporal resolution. These techniques could reveal previously unidentified TRIM71 interaction partners in different cellular compartments and under various biological contexts, providing insights into its multifaceted regulatory roles .

Advanced microscopy techniques like lattice light-sheet microscopy using FITC-conjugated TRIM71 antibodies could enable long-term imaging of TRIM71 dynamics in living cells with minimal phototoxicity. This approach would be particularly valuable for tracking TRIM71's involvement in dynamic processes such as cell division or stress responses in stem cells or cancer models . Similarly, super-resolution microscopy methods such as DNA-PAINT, which offers exceptional multiplexing capabilities, could visualize TRIM71's co-localization with multiple binding partners simultaneously at nanoscale resolution.

CRISPR-based techniques for visualizing endogenous TRIM71, such as CRISPR-Sirius, combined with antibody validation, provide opportunities to study TRIM71 dynamics without overexpression artifacts. Additionally, integrating TRIM71 antibody-based detection with spatial transcriptomics and proteomics would create comprehensive maps of how TRIM71 expression relates to local gene expression patterns within tissues or tumor microenvironments .

The development of bi-specific antibodies or antibody-drug conjugates targeting TRIM71 could enable novel therapeutic approaches for cancers where TRIM71 is overexpressed . Similarly, engineered antibody fragments (Fabs or nanobodies) against TRIM71 could serve as research tools for disrupting specific protein-protein interactions without affecting the entire TRIM71 complex, allowing for more precise functional studies of TRIM71's different domains and interactions .

How might single-cell analysis techniques utilizing TRIM71 antibodies advance our understanding of cellular heterogeneity?

Single-cell analysis techniques incorporating TRIM71 antibodies offer transformative potential for understanding cellular heterogeneity in development, homeostasis, and disease. Mass cytometry (CyTOF) using metal-conjugated TRIM71 antibodies enables high-dimensional phenotyping of cells based on TRIM71 expression alongside dozens of other markers. This approach could reveal previously unrecognized cell subpopulations with distinct TRIM71 expression levels and correlate these with stemness markers, cell cycle status, or differentiation markers in heterogeneous tissues or tumors .

Single-cell resolution imaging using multiplexed immunofluorescence with FITC-conjugated TRIM71 antibodies, combined with cyclic immunofluorescence or CO-Detection by indEXing (CODEX), allows visualization of TRIM71 expression patterns across entire tissue sections while preserving spatial relationships. This methodology is particularly valuable for understanding the tissue microenvironment influences on TRIM71 expression and function, especially in contexts like hepatocellular carcinoma where TRIM71 appears to play an oncogenic role .

Integration of TRIM71 antibody-based sorting with single-cell RNA sequencing creates powerful platforms for correlating TRIM71 protein levels with transcriptomic profiles at the single-cell level. This approach could identify gene expression signatures associated with varying TRIM71 levels and potentially discover new TRIM71-regulated pathways. Additionally, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) using oligo-conjugated TRIM71 antibodies would allow simultaneous measurement of TRIM71 protein expression and transcriptome-wide gene expression in thousands of individual cells.

For developmental biology applications, trajectory inference analyses based on TRIM71 expression in single cells could map how TRIM71 levels change during differentiation processes and identify branch points where TRIM71 might influence cell fate decisions . Similar approaches applied to cancer models could reveal how TRIM71 expression heterogeneity contributes to tumor evolution, therapeutic resistance, or metastatic potential . Furthermore, single-cell multi-omics approaches that combine measurements of TRIM71 protein levels with chromatin accessibility, DNA methylation, or metabolic profiles would provide unprecedented insights into the mechanisms through which TRIM71 regulates cellular identity and function.

What future research directions might TRIM71 antibodies facilitate in understanding disease mechanisms?

TRIM71 antibodies will likely facilitate several promising research directions for understanding disease mechanisms. In neurodevelopmental disorders, where TRIM71's role in neural progenitor cell maintenance is critical , FITC-conjugated TRIM71 antibodies could enable detailed mapping of expression patterns in brain organoids derived from patient iPSCs. This approach would help elucidate how alterations in TRIM71 expression or function contribute to neurodevelopmental pathologies, potentially revealing new therapeutic targets.

In reproductive medicine, TRIM71 antibodies can advance our understanding of infertility mechanisms, given the recently established association between TRIM71 deficiency and male infertility . Immunohistochemical analysis of testicular biopsies from infertile patients using TRIM71 antibodies could identify subgroups with TRIM71-related pathology, potentially leading to more targeted therapeutic approaches. Similarly, studying TRIM71's expression and localization during gametogenesis using high-resolution imaging with FITC-conjugated antibodies might reveal critical stages where TRIM71 dysfunction contributes to reproductive disorders.

For oncology research, TRIM71 antibodies will enable comprehensive characterization of TRIM71's role in hepatocellular carcinoma and other cancers where it appears to drive carcinogenesis . Prognostic studies correlating TRIM71 expression patterns with patient outcomes could establish TRIM71 as a clinically relevant biomarker. Furthermore, mechanistic investigations using TRIM71 antibodies to identify cancer-specific interaction partners might reveal vulnerabilities that could be therapeutically targeted. The development of therapeutic antibodies or antibody-drug conjugates targeting TRIM71 represents an exciting frontier for treating cancers with TRIM71 overexpression.

In stem cell biology and regenerative medicine, TRIM71 antibodies will continue to be instrumental in understanding how TRIM71 regulates the balance between self-renewal and differentiation . This knowledge could inform strategies for improving the efficiency and safety of stem cell-based therapies. Additionally, investigating TRIM71's interaction with the lncRNA Trincr1 and its effects on FGF/ERK signaling using combined RNA-protein detection approaches might reveal new methods for manipulating stem cell behavior in therapeutic contexts .

Finally, in immunology and inflammation research, exploring TRIM71's potential roles using antibody-based detection methods could uncover previously unrecognized functions in immune cell development or inflammatory responses, given the importance of related TRIM family proteins in immunity and the known roles of miRNAs like let-7 in immune regulation .