TRUB1 Antibody

What is TRUB1 and what cellular functions does it perform?

TRUB1 (TruB pseudouridine synthase homolog 1) is a 349-amino acid protein that functions as a pseudouridine synthase responsible for converting uridine to pseudouridine in RNA molecules. The protein contains a characteristic VFAVHKPKGPTSA box in positions 71-83, which corresponds to motif I of the TruB family and is likely involved in maintaining protein structure . TRUB1 is primarily known for catalyzing pseudouridylation in both non-coding RNAs and messenger RNAs, with substantial evidence identifying it as the predominant pseudouridine synthase acting on mammalian mRNAs . Recent research has revealed that TRUB1 plays crucial roles beyond tRNA modification, including regulation of microRNA processing through direct binding to primary miRNAs such as let-7 . Additionally, TRUB1-mediated pseudouridylation has been shown to enhance mRNA stability, suggesting a post-transcriptional regulatory function . This multifaceted protein represents an important component of the RNA modification machinery that influences gene expression patterns in mammalian cells.

What applications is the TRUB1 antibody validated for?

The TRUB1 antibody (such as the 12520-1-AP polyclonal antibody) has been validated for multiple experimental applications essential for studying this protein's expression and function. It is specifically validated for Western Blot (WB) applications with confirmed detection in HEK-293T and HepG2 cells at a recommended dilution range of 1:1000-1:4000 . For Immunoprecipitation (IP), the antibody has been validated using mouse liver tissue with a recommended usage of 0.5-4.0 μg for every 1.0-3.0 mg of total protein lysate . In Immunohistochemistry (IHC) applications, it successfully detects TRUB1 in human kidney tissue and mouse testis tissue at dilutions of 1:50-1:500, with optimal antigen retrieval using TE buffer at pH 9.0 (alternatively, citrate buffer at pH 6.0 can be used) . The antibody has also been validated for Immunofluorescence (IF) as documented in published literature . Additionally, while not extensively detailed in the search results, the antibody is reported suitable for ELISA applications. When designing experiments, researchers should consider that this antibody demonstrates validated reactivity with human and mouse samples, making it versatile for comparative studies across these species.

What is the recommended protocol for detecting TRUB1 by Western blot?

For optimal detection of TRUB1 by Western blot using the 12520-1-AP antibody, researchers should follow a methodological approach that accounts for the protein's characteristics. Begin by preparing protein lysates from appropriate samples such as HEK-293T or HepG2 cells, which have been confirmed to express detectable levels of TRUB1 . Separation should be performed using standard SDS-PAGE conditions with particular attention to resolving the region around 37 kDa, which is the observed molecular weight of TRUB1 . After protein transfer to an appropriate membrane, blocking should be performed according to standard protocols. For primary antibody incubation, dilute the TRUB1 antibody within the recommended range of 1:1000-1:4000 in appropriate antibody dilution buffer . The antibody concentration should be optimized for each experimental system, as recommended in the product information. For visualization, use an appropriate secondary anti-rabbit IgG antibody compatible with your detection system. When interpreting results, expect to observe a band at approximately 37 kDa, corresponding to the calculated molecular weight of TRUB1 . For more challenging samples or when signal optimization is needed, researchers may need to adjust the primary antibody concentration within the recommended range or modify incubation conditions to achieve optimal signal-to-noise ratio.

How can TRUB1 antibody be used to investigate RNA modification mechanisms?

TRUB1 antibody can be strategically employed to investigate RNA modification mechanisms through several sophisticated approaches. For comprehensive mapping of TRUB1-dependent pseudouridylation sites, researchers can implement RNA immunoprecipitation (RIP) combined with high-throughput sequencing. This methodology has successfully revealed direct interactions between TRUB1 and its RNA targets, including both tRNAs and primary microRNAs like pri-let-7a1 . To execute this approach, immunoprecipitate TRUB1-RNA complexes using the validated antibody (0.5-4.0 μg per 1.0-3.0 mg of total protein lysate), followed by RNA extraction and sequencing . For more precise identification of direct binding sites, high-throughput crosslinking immunoprecipitation (HITS-CLIP) can be employed as demonstrated in previous studies where a 3xFLAG-tagged TRUB1 was used to map interactions across the transcriptome . This technique revealed that beyond tRNAs, TRUB1 interacts with mRNAs, lncRNAs, and miRNAs . For functional studies examining the impact of TRUB1-mediated pseudouridylation on RNA stability, researchers can implement TRUB1 knockdown followed by RNA-seq analysis of transcript half-lives, which has revealed that TRUB1 targets display shorter half-lives upon TRUB1 depletion . Additionally, to validate pseudouridylation sites mapped on viral and cellular transcripts, researchers can use TRUB1 knockout cell lines in conjunction with techniques like PA-Ψ-seq, which has been successfully employed to identify TRUB1-dependent pseudouridine modifications .

What approaches can detect TRUB1 interactions with other RNA processing factors?

To investigate TRUB1's interactions with other RNA processing factors, researchers can employ several sophisticated methodological approaches. Co-immunoprecipitation (Co-IP) using TRUB1 antibody has successfully demonstrated physical interactions between TRUB1 and RNA processing factors such as DGCR8, a component of the microprocessor complex involved in miRNA processing . For this approach, immunoprecipitation with anti-TRUB1 antibody followed by immunoblotting for suspected interacting partners can reveal protein-protein associations. Reciprocal Co-IP (e.g., immunoprecipitating with anti-DGCR8 followed by immunoblotting with anti-TRUB1) should be performed to confirm these interactions . For investigating the functional relevance of these interactions, researchers can combine TRUB1 knockdown with RIP assays targeting RNA processing factors like DGCR8, which has revealed that TRUB1 depletion reduces interactions between DGCR8 and pri-let-7a1, suggesting that TRUB1 enhances complex formation between RNA processing factors and their RNA targets . To study competitive binding dynamics, as demonstrated with Lin28B and TRUB1 for pri-let-7a1 binding, RIP assays can be performed in cells with knockdown of competing factors to assess how relative binding affinities change . For a more comprehensive identification of the TRUB1 interactome, proximity-based labeling techniques followed by mass spectrometry analysis could be implemented, though this approach would require fusion of TRUB1 with a proximity labeling enzyme.

How should researchers interpret TRUB1 antibody signals in cancer cell studies?

Interpreting TRUB1 antibody signals in cancer cell studies requires careful consideration of several factors related to TRUB1's functional roles in cancer biology. Recent research has revealed that TRUB1-mediated pseudouridylation contributes to transcript stabilization in human cancer cells, with TRUB1 knockdown leading to reduced half-life of target mRNAs . When analyzing Western blot data from cancer cell lines, researchers should compare TRUB1 expression levels against appropriate controls, considering that altered TRUB1 expression may reflect changes in RNA modification requirements in different cancer types. Quantification of expression differences should be performed using standardized methods, with normalization to housekeeping proteins appropriate for the specific cancer cell type being studied. For immunohistochemistry applications in cancer tissues, signals should be interpreted with consideration of cancer-specific protein localization patterns, as TRUB1's subcellular distribution may provide insights into its functional state . Additionally, when investigating correlations between TRUB1 expression and cancer phenotypes, researchers should consider examining the stability and expression levels of known TRUB1 targets, including cancer-relevant transcripts like ERH, SCP2, AMFR and CDC6, which have been shown to be regulated by TRUB1-dependent pseudouridylation . The following table summarizes key TRUB1 targets in cancer research and their response to TRUB1 depletion:

What are the optimal conditions for performing TRUB1 immunoprecipitation experiments?

For optimal TRUB1 immunoprecipitation experiments, researchers should implement a methodical approach that maximizes specificity and yield. Based on validated protocols, immunoprecipitation should be performed using 0.5-4.0 μg of TRUB1 antibody for every 1.0-3.0 mg of total protein lysate . When preparing lysates from tissues, mouse liver tissue has been specifically validated for successful TRUB1 immunoprecipitation . The lysis buffer composition should maintain protein-protein and protein-RNA interactions if studying TRUB1 complexes; typical buffers contain 1× PBS with detergents such as 0.1% SDS, 0.5% DOC, and 0.5% NP-40 . For RNA immunoprecipitation (RIP) applications, samples may require treatment with RNase inhibitors to preserve RNA integrity, and DNase treatment to eliminate DNA contamination. Washing conditions should be stringent enough to reduce non-specific binding while preserving specific interactions; validated protocols have successfully used wash buffer (1× PBS, 0.1% SDS, 0.5% DOC, 0.5% NP-40) followed by high-salt-wash buffer (5× PBS, 0.1% SDS, 0.5% DOC, 0.5% NP-40) . For elution, standard protocols using SDS sample buffer are appropriate for protein analysis, while for RNA-protein complex studies, proteinase K treatment has been successfully employed . When designing immunoprecipitation experiments to study specific TRUB1 interactions, such as with DGCR8 or Lin28B, optimization of crosslinking conditions might be necessary to capture transient interactions . For validation of immunoprecipitation specificity, researchers should include appropriate controls such as IgG control antibodies and, when possible, TRUB1 knockout or knockdown samples.

What techniques can detect pseudouridylation sites regulated by TRUB1?

Several sophisticated techniques can be employed to detect and validate pseudouridylation sites regulated by TRUB1. BID-seq (Biochemical Intracellular Detection of Ψ sequencing) has emerged as a powerful method that identifies pseudouridylation sites based on deletion signatures generated during reverse transcription at Ψ-BS (pseudouridine-bisulfite) sites . This technique can detect pseudouridylation with high efficiency, inducing more than 50% deletion rates at Ψ sites in various sequence contexts, with very low background deletion rates at untreated sites . For validation of TRUB1-specific pseudouridylation sites, genetic approaches using TRUB1 knockdown or knockout followed by Ψ-mapping techniques have proven effective . These approaches have revealed that TRUB1 is responsible for pseudouridylation at approximately 60% of high-confidence pseudouridylation sites in human cells . PA-Ψ-seq (Photo-Activatable Ribonucleoside-Enhanced Crosslinking and Pseudouridine site identification) has been used to map pseudouridine residues on viral and cellular transcripts, allowing comparison between wild-type and TRUB1 knockout cells to identify TRUB1-dependent modifications . CMC (N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate)-based RT stop methods can be used to validate specific pseudouridylation sites, particularly when multiple uridines are adjacent to each other . For functional validation of TRUB1-regulated pseudouridylation, engineered systems using dCas13d-TRUB1 fusion proteins have been developed to install site-specific pseudouridine modifications, allowing researchers to directly test the impact of pseudouridylation on transcript stability and other functions .

How can researchers troubleshoot non-specific signals when using TRUB1 antibody?

When encountering non-specific signals with TRUB1 antibody, researchers should implement a systematic troubleshooting approach. First, optimize antibody concentration by testing dilutions across the recommended range (1:1000-1:4000 for Western blot and 1:50-1:500 for IHC) to identify the optimal signal-to-noise ratio for your specific experimental system . Increasing the stringency of washing steps can significantly reduce background; for Western blots, consider longer or additional washes with TBS-T or PBS-T, while for immunohistochemistry, optimize washing steps between primary and secondary antibody incubations. Blocking conditions should be carefully adjusted; insufficient blocking often leads to high background, so experiment with different blocking agents (BSA, non-fat dry milk, or commercial blocking reagents) and durations to identify optimal conditions. For immunohistochemistry applications, antigen retrieval methods significantly impact specificity; the TRUB1 antibody documentation specifically recommends TE buffer at pH 9.0, with citrate buffer at pH 6.0 as an alternative . Compare these methods to determine which provides the best signal-to-noise ratio for your tissue type. To confirm signal specificity, incorporate appropriate controls including TRUB1 knockdown or knockout samples when available. If multiple bands appear in Western blots, consider that post-translational modifications, proteolytic degradation, or alternative splicing variants might be present; tissue or cell type-specific expression patterns should be considered when interpreting results. For persistent non-specific binding issues, pre-absorption of the antibody with the immunizing peptide (if available) can be used as a control to identify specific versus non-specific signals.

What factors influence the detection of TRUB1-dependent RNA modifications?

Multiple factors influence the successful detection of TRUB1-dependent RNA modifications, requiring careful experimental design and execution. RNA quality and integrity are paramount; degraded RNA can lead to false negatives or positives in modification detection assays. Implement rigorous RNA extraction protocols that minimize RNase contamination and include integrity checks via Bioanalyzer or gel electrophoresis before proceeding with modification detection. The sensitivity of detection methods varies significantly; BID-seq has demonstrated high efficiency with more than 50% deletion rates at Ψ sites in various sequence contexts, but requires sufficient read coverage for detecting low-abundance modifications . Read depth is particularly critical for low-expressed transcripts, as detection of pseudouridine modifications requires sufficient coverage to distinguish modification-induced signatures from background . Sequence context surrounding the modification site impacts detection efficiency; when a pseudouridine site is adjacent to one or more uridines, determining the exact position can be challenging using deletion-based methods alone, necessitating complementary approaches like CMC-based RT stop . The specificity of the pseudouridylation signature is another critical factor; some sequence motifs (NNUNN) show background deletion rates of 10-25% after BID-seq treatment, requiring stringent analysis criteria to eliminate false positives . For TRUB1-specific modifications, effective knockdown or knockout verification is essential; incomplete depletion can lead to residual pseudouridylation and confound results . Additionally, the subcellular compartmentalization of RNA can affect modification patterns; consider fraction-specific analyses to comprehensively map modifications in different cellular compartments.

How can researchers validate the specificity of TRUB1 antibody in their experimental system?

Validating the specificity of TRUB1 antibody in a particular experimental system requires a multi-faceted approach. The gold standard for antibody validation is testing in TRUB1 knockout or knockdown systems, where the specific signal should be significantly reduced or eliminated compared to controls. This approach has been successfully implemented in studies examining TRUB1-dependent pseudouridylation . For cell line validation, Western blot analysis should show a predominant band at the expected molecular weight of 37 kDa, which aligns with the calculated molecular weight of the 349-amino acid TRUB1 protein . When validating in new cell types or tissues beyond the confirmed HEK-293T cells, HepG2 cells, human kidney tissue, and mouse testis tissue, perform comparative analyses with these validated positive controls . Peptide competition assays provide another layer of validation; pre-incubating the antibody with excess immunizing peptide should substantially reduce or eliminate specific binding signals. For RNA immunoprecipitation applications, validation should include demonstration that known TRUB1 RNA targets are enriched in TRUB1 immunoprecipitates compared to control immunoprecipitates; tRNAs serve as excellent positive controls as they are well-established TRUB1 substrates . Cross-reactivity testing with related proteins, particularly other pseudouridine synthases, can provide additional evidence of specificity. When possible, validate results using an alternative TRUB1 antibody with a different epitope to confirm signal specificity. For immunohistochemistry applications, correlation with mRNA expression data (e.g., from public databases) can provide supporting evidence for antibody specificity across tissues with varying TRUB1 expression levels.

How can TRUB1 antibody be used to investigate its role in miRNA processing?

TRUB1 antibody can be instrumental in investigating its recently discovered role in miRNA processing through several methodological approaches. RNA immunoprecipitation (RIP) assays using TRUB1 antibody have revealed direct binding between TRUB1 and primary miRNAs, particularly pri-let-7a1, demonstrating that TRUB1 physically interacts with pri-miRNAs as part of their processing pathway . Researchers can implement this approach using 0.5-4.0 μg of TRUB1 antibody per 1.0-3.0 mg of protein lysate, followed by RNA extraction and qPCR for specific miRNAs of interest . To explore the mechanism by which TRUB1 influences miRNA processing, co-immunoprecipitation experiments can be performed to examine interactions between TRUB1 and components of the microprocessor complex, such as DGCR8 . Previous research has demonstrated that TRUB1 knockdown reduces the interaction between pri-let-7a1 and DGCR8, suggesting that TRUB1 enhances complex formation between DGCR8 and primary miRNAs . For a more comprehensive understanding of TRUB1's impact on the miRNA landscape, researchers can combine TRUB1 knockdown with miRNA profiling technologies like TaqMan arrays, which previously identified miR-29b, miR-139, and miR-107 as miRNAs whose mature forms decrease upon TRUB1 depletion . To investigate potential competition between TRUB1 and other miRNA regulatory factors, such as the documented competition with Lin28B for binding to pri-let-7a1, competitive binding assays can be designed where one factor is depleted and the binding of the other is assessed using RIP . For dissecting the structural basis of TRUB1 recognition of miRNA precursors, in vitro binding assays like EMSA using recombinant TRUB1 protein and mutant RNA constructs with modified loop structures can reveal specific recognition elements, as demonstrated by the loss of binding observed with a loop mutant of pri-let-7a1 .

What is the significance of TRUB1-mediated pseudouridylation in mRNA stability?

TRUB1-mediated pseudouridylation plays a critical role in mRNA stability, representing a significant post-transcriptional regulatory mechanism in gene expression control. Recent research has demonstrated that TRUB1 is a predominant pseudouridine synthase acting on mammalian mRNAs, responsible for pseudouridylation at approximately 60% of high-confidence pseudouridylation sites in human cells . Functional studies have revealed that TRUB1-dependent pseudouridylation directly impacts transcript half-life. When TRUB1 is knocked down, target transcripts containing TRUB1-modified pseudouridines display shortened half-lives compared to non-target mRNAs without detectable pseudouridine modifications . This stabilizing effect has been validated through both loss-of-function and gain-of-function approaches. In knockdown experiments, representative TRUB1 targets such as ERH, SCP2, AMFR, and CDC6 showed reduced mRNA levels after siTRUB1 treatment, with subsequent validation confirming reduced stability for all four targets but not for non-target mRNAs . Conversely, engineered site-specific pseudouridine deposition using a fused dCas13d-TRUB1 system successfully prolonged mRNA lifetime, providing direct evidence that pseudouridylation installation can enhance transcript stability . This stability effect represents a counterpoint to another abundant mRNA modification, m6A, which tends to destabilize mRNAs, suggesting that different RNA modifications may have opposing effects on transcript fate . Additionally, TRUB1-dependent pseudouridylation has been observed at mRNA stop codons, with potential implications for stop codon readthrough in specific tissues, suggesting further functional consequences beyond stability regulation .

How can researchers integrate TRUB1 antibody in the study of engineered RNA modification systems?

Researchers can strategically integrate TRUB1 antibody in the study of engineered RNA modification systems through multiple innovative approaches. The development of fusion proteins combining TRUB1 with RNA-targeting modules, such as the dCas13d-TRUB1 system, represents a significant advancement in site-directed pseudouridylation technology . TRUB1 antibody can be employed to validate the expression and stability of such fusion proteins through Western blot analysis, ensuring that the engineered constructs maintain appropriate expression levels in the experimental system. For direct validation of fusion protein function, TRUB1 antibody can be used in RNA immunoprecipitation (RIP) experiments to confirm that the engineered TRUB1 fusion maintains RNA binding capability and to identify RNA targets of the fusion protein . When developing inducible or tissue-specific TRUB1 modification systems, the antibody can be used to monitor expression levels and localization of the engineered construct across different conditions or tissues using techniques like Western blot, immunohistochemistry, or immunofluorescence . To investigate potential differences in the RNA substrate specificity between native TRUB1 and engineered TRUB1 fusions, comparative RIP-seq experiments can be performed using the antibody to immunoprecipitate both the native and engineered forms, followed by high-throughput sequencing to map their respective binding sites . For functional validation of engineered systems, researchers can implement HITS-CLIP or similar techniques using TRUB1 antibody to map the binding sites of TRUB1 fusion proteins genome-wide and correlate these with pseudouridylation sites detected using techniques like BID-seq . Additionally, co-immunoprecipitation experiments using TRUB1 antibody can reveal how fusion proteins interact with endogenous RNA processing machinery, providing insights into the integration of engineered modification systems with native cellular processes .

What are emerging applications of TRUB1 antibody in disease-related research?

Emerging applications of TRUB1 antibody in disease-related research span multiple areas where RNA modification dysregulation contributes to pathological processes. In cancer research, TRUB1 antibody can be used to investigate aberrant pseudouridylation patterns, as evidence suggests TRUB1-mediated pseudouridylation plays a role in transcript stabilization of cancer-relevant genes . Immunohistochemistry and tissue microarray applications can reveal TRUB1 expression patterns across different cancer types and stages, potentially identifying diagnostic or prognostic biomarkers . For investigating TRUB1's role in disease-associated microRNA dysregulation, the antibody can be employed in RIP experiments to examine altered binding patterns between TRUB1 and oncogenic or tumor-suppressive miRNAs, building upon established connections between TRUB1 and miRNA processing pathways . In the context of potential therapeutic applications, TRUB1 antibody can validate knockdown efficiency in studies exploring TRUB1 inhibition as a means to modulate stability of oncogenic transcripts . Conversely, for genetic diseases involving premature stop codons, the antibody can help validate engineered TRUB1-based systems designed to induce stop codon readthrough, as pseudouridylation at stop codons has been implicated in this process . For viral infection research, TRUB1 antibody can aid in studying host-pathogen interactions involving RNA modification, building on existing work mapping pseudouridine residues on viral transcripts . In comparative pathology, the antibody's validated reactivity in both human and mouse samples enables translational studies between mouse models and human disease states . Furthermore, as pseudouridylation is increasingly recognized as a regulator of innate immune response, TRUB1 antibody can facilitate investigation of connections between dysregulated RNA modification and autoimmune or inflammatory conditions.