trnau1ap Antibody

What is TRNAU1AP and what cellular functions does it regulate?

TRNAU1AP, originally named SECp43, is a highly conserved 43-kDa tRNA[Ser]Sec-binding protein identified through affinity purification. It plays a key role in selenoprotein biosynthesis by facilitating several critical interactions in the selenium incorporation pathway. TRNAU1AP interacts with the selenocysteyl-tRNA[Ser]Sec-Sec-specific elongation factor (EFsec) complex in vitro and co-expression of TRNAU1AP facilitates interaction between EFsec and SBP2 in vivo. Additionally, TRNAU1AP mediates Sec incorporation and upregulates selenoprotein mRNA expression levels .

Research has demonstrated that TRNAU1AP influences the expression of multiple selenoproteins, including glutathione peroxidase 1 (GPx1), thioredoxin reductase 1 (Txnrd1), and selenoprotein K (SelK). Experimental downregulation of TRNAU1AP resulted in extensive reduction of selenoprotein expression, highlighting its crucial role in selenoprotein synthesis machinery .

What applications is the TRNAU1AP antibody validated for?

The TRNAU1AP/SECP43 antibody (15053-1-AP) has been validated for multiple research applications:

The antibody has demonstrated positive Western blot detection in multiple cell lines including A549, Jurkat, MCF-7, and COLO 320 cells. For immunohistochemistry, it has been validated with human hepatocirrhosis tissue, with recommended antigen retrieval using TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0 .

Additionally, this antibody has been used in knockdown/knockout studies as referenced in published literature, making it suitable for validating gene manipulation experiments .

What is the molecular weight of TRNAU1AP protein and what should I expect on Western blots?

When using the TRNAU1AP antibody in Western blot applications, researchers should note that while the calculated molecular weight of the protein is 32 kDa, the observed molecular weights are typically 36 kDa and 21 kDa . This discrepancy between calculated and observed weights is important to consider when analyzing Western blot results and may reflect post-translational modifications or alternative splicing events.

Proper positive controls should be run alongside experimental samples, with recommended cell lines including A549, Jurkat, MCF-7, or COLO 320 cells, which have been validated to express detectable levels of TRNAU1AP .

What are the optimal protocols for using TRNAU1AP antibody in Western blot applications?

For optimal Western blot results with TRNAU1AP antibody, the following methodology is recommended:

• Sample preparation: Prepare protein lysates from appropriate cell lines (A549, Jurkat, MCF-7, or COLO 320 cells work well as positive controls).

• Antibody dilution: Use the TRNAU1AP antibody at a dilution range of 1:500-1:2000, optimizing for your specific experimental system .

• Detection strategy: The antibody should be used according to standard Western blot protocols with appropriate secondary antibodies.

• Expected bands: Look for primary bands at approximately 36 kDa and 21 kDa, not at the calculated 32 kDa .

• Controls: Always include positive control samples from validated cell lines and negative controls such as knockdown samples when available.

It is recommended that researchers titrate this reagent in each testing system to obtain optimal results, as sample-dependent variations may occur .

How should TRNAU1AP antibody be used for immunohistochemistry applications?

For immunohistochemistry applications, the following protocol considerations are important:

• Tissue preparation: Properly fixed and processed tissue sections (paraffin-embedded or frozen) should be used.

• Antigen retrieval: Recommended antigen retrieval should be performed with TE buffer pH 9.0, although citrate buffer pH 6.0 may be used as an alternative .

• Antibody dilution: Use the antibody at a dilution range of 1:20-1:200, optimizing based on your specific tissue type and detection system .

• Positive control tissue: Human hepatocirrhosis tissue has been validated as a positive control for IHC applications .

• Detection system: Use an appropriate secondary antibody and detection system compatible with rabbit IgG primary antibodies.

• Counterstaining: Apply standard counterstaining protocols to visualize tissue architecture alongside the specific antibody staining.

What are the storage and handling recommendations for maintaining TRNAU1AP antibody activity?

To maintain optimal activity of TRNAU1AP antibody:

• Storage temperature: Store the antibody at -20°C where it remains stable for one year after shipment .

• Buffer composition: The antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting: Aliquoting is unnecessary for -20°C storage, although it may be beneficial for frequently used antibodies to avoid freeze-thaw cycles .

• BSA content: Small volume sizes (20μl) contain 0.1% BSA .

• Working solution handling: Keep on ice when in use and avoid repeated freeze-thaw cycles.

How can TRNAU1AP knockdown experiments be designed to study selenoprotein functions?

Designing effective TRNAU1AP knockdown experiments requires careful consideration of several factors:

• siRNA selection: Testing multiple siRNAs targeting TRNAU1AP is recommended. Research has shown that some siRNAs (like siRNA-1 in published studies) may be more effective than others. RT-qPCR should be performed 24 hours post-transfection to confirm knockdown efficiency .

• Protein knockdown verification: Western blotting should be performed approximately 48 hours post-transfection to confirm underexpression of TRNAU1AP at the protein level .

• Downstream selenoprotein assessment: Following TRNAU1AP knockdown, it is important to assess the expression levels of various selenoproteins such as GPx1, Txnrd1, and SelK. Published research demonstrates that modulating TRNAU1AP expression levels affects the expression of these selenoproteins .

• Functional assays: Cell proliferation can be assessed using MTT assays and by measuring proliferating cell nuclear antigen (PCNA) expression levels. Research has shown that TRNAU1AP knockdown decreases cell proliferation compared to control groups .

• Apoptosis assessment: Annexin V-FITC and PI double staining followed by flow cytometric analysis can be used to measure apoptotic rates. Research indicates that TRNAU1AP siRNA-transfected groups show increased apoptotic rates compared to control groups .

What methodological approaches can be used to study TRNAU1AP's role in selenoprotein biosynthesis?

To investigate TRNAU1AP's role in selenoprotein biosynthesis, researchers can employ several methodological approaches:

• Protein interaction studies: Co-immunoprecipitation experiments can be used to study interactions between TRNAU1AP and other components of the selenoprotein biosynthesis machinery, such as EFsec and SBP2 .

• Subcellular localization: Immunofluorescence microscopy can be used to examine the co-localization of TRNAU1AP with SepSecS and to investigate how their interactions affect cellular localization. Research has shown that when SepSecS and TRNAU1AP are co-expressed, TRNAU1AP induces SepSecS to localize to the cytoplasm rather than the nucleus .

• Selenoprotein expression profiling: Following TRNAU1AP manipulation, comprehensive analysis of selenoprotein expression can be performed using Western blotting, qPCR, or proteomics approaches .

• Selenium supplementation studies: Investigating the effects of selenium availability on TRNAU1AP function can provide insights into its role in selenium metabolism. Given that selenium deficiency results in various cardiovascular diseases including Keshan disease, studying TRNAU1AP in cardiomyocyte models under different selenium conditions may be particularly relevant .

How can TRNAU1AP antibody be used to investigate selenium deficiency-related pathologies?

TRNAU1AP antibody can be instrumental in investigating selenium deficiency-related pathologies through several experimental approaches:

• Tissue expression analysis: IHC staining of tissues from selenium-deficient models can reveal changes in TRNAU1AP expression patterns. Human hepatocirrhosis tissue has been validated for TRNAU1AP antibody in IHC applications .

• Cardiomyopathy models: Given the association between selenium deficiency and Keshan disease (characterized by heart failure and severe cardiomyopathy), TRNAU1AP expression and localization in cardiac tissues can be studied using appropriate antibody applications. H9c2 cardiomyocyte cell lines have been used in previous TRNAU1AP studies .

• Oxidative stress assessment: Selenium deficiency results in reactive oxygen species accumulation and myocardial injury. TRNAU1AP antibody can be used to correlate TRNAU1AP expression with markers of oxidative stress and selenoprotein expression (particularly GPx1 and Txnrd1) in experimental models .

• Intervention studies: The antibody can be used to monitor changes in TRNAU1AP expression following selenium supplementation or other interventions in deficiency models.

What are common issues when using TRNAU1AP antibody in Western blot applications and how can they be addressed?

When using TRNAU1AP antibody in Western blot applications, researchers may encounter several common issues:

• Multiple bands: Aside from the expected bands at 36 kDa and 21 kDa, additional bands might appear. This could be due to:

  • Post-translational modifications

  • Alternative splicing variants

  • Protein degradation

  • Non-specific binding

  Solution: Include positive control lysates from validated cell lines (A549, Jurkat, MCF-7, or COLO 320) alongside experimental samples. Optimize antibody dilution within the recommended range (1:500-1:2000) .

• Weak signal: If the TRNAU1AP signal is faint or undetectable:

  • Increase protein loading amount

  • Reduce antibody dilution (use more concentrated antibody)

  • Extend primary antibody incubation time

  • Use more sensitive detection reagents

  • Verify that your samples express TRNAU1AP (not all cell types express it at equal levels)

• High background: If experiencing excessive background:

  • Increase blocking time or change blocking reagent

  • Increase washing steps duration and frequency

  • Further dilute the primary antibody

  • Reduce exposure time during detection

How can researchers validate the specificity of TRNAU1AP antibody in their experimental systems?

Validating antibody specificity is crucial for reliable research outcomes. For TRNAU1AP antibody, consider these validation approaches:

• Knockdown/knockout controls: Utilize RNA interference (siRNA) to knockdown TRNAU1AP expression. Published protocols have successfully used siRNA targeting TRNAU1AP, with siRNA-1 showing particular effectiveness in reducing TRNAU1AP expression, which can be confirmed by RT-qPCR after 24 hours and Western blotting after 48 hours .

• Overexpression controls: Compare antibody signal in cells transfected with TRNAU1AP expression vectors versus mock-transfected cells. Previous research has utilized pcDNA 3.1(+)-TRNAU1AP for overexpression studies .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to demonstrate binding specificity.

• Cross-species reactivity: The TRNAU1AP antibody has been tested for reactivity with human, mouse, and rat samples , making it useful for comparative studies across these species.

• Multiple detection methods: Validate findings using different antibody-based techniques (Western blot, IHC, immunofluorescence) to confirm consistent protein detection.

What factors should be considered when interpreting TRNAU1AP expression data in different experimental contexts?

When interpreting TRNAU1AP expression data, several factors should be considered:

• Expression in different cell types: TRNAU1AP expression varies across cell types. The antibody has been validated in several cell lines including A549, Jurkat, MCF-7, and COLO 320 cells , which can serve as reference points.

• Correlation with selenoprotein expression: Changes in TRNAU1AP expression should be interpreted alongside changes in selenoprotein levels. Research has shown that modulating TRNAU1AP expression affects GPx1, Txnrd1, and SelK expression levels .

• Selenium status: Selenium availability can influence the selenoprotein biosynthesis pathway. Consider measuring selenium levels or supplementing experimental systems with selenium when studying TRNAU1AP function.

• Cellular processes affected: TRNAU1AP impacts fundamental cellular processes:

  • Cell proliferation (can be assessed by MTT assay and PCNA expression)

  • Apoptosis (can be assessed by Annexin V-FITC and PI double staining)
    Research has shown that TRNAU1AP knockdown decreases proliferation and increases apoptosis .

• Subcellular localization: TRNAU1AP's function may depend on its cellular location and interactions. It has been shown to affect the localization of SepSecS, promoting its cytoplasmic rather than nuclear localization when co-expressed .

How is TRNAU1AP being studied in cardiovascular disease models?

TRNAU1AP is emerging as an important factor in cardiovascular research, particularly in relation to selenium deficiency-induced pathologies:

• Keshan disease connection: Selenium deficiency results in Keshan disease, a cardiomyopathy characterized by heart failure and severe cardiomyopathy with arrhythmia and congestive heart failure . TRNAU1AP, as a key player in selenoprotein biosynthesis, is being investigated in this context.

• Cardiomyocyte models: H9c2 cardiomyocyte cells have been used to study TRNAU1AP function through both knockdown and overexpression approaches. Research has demonstrated that modulating TRNAU1AP expression affects cardiomyocyte proliferation and apoptosis .

• Selenoprotein regulation: In cardiac contexts, TRNAU1AP has been shown to affect the expression of selenoproteins implicated in cardiac protection against oxidative stress, including GPx1 and Txnrd1 .

• Reactive oxygen species accumulation: Previous studies have demonstrated that selenium deficiency leads to reactive oxygen species accumulation and myocardial injury in mouse models . TRNAU1AP's role in this process is being actively investigated.

What is known about the structural determinants of TRNAU1AP's function in selenoprotein synthesis?

The structural basis of TRNAU1AP function is an area of ongoing research:

• tRNA[Ser]Sec binding: TRNAU1AP was originally identified as a 43-kDa tRNA[Ser]Sec-binding protein . The structural elements that mediate this interaction are not fully characterized but are critical to its function.

• Protein-protein interactions: TRNAU1AP interacts with the selenocysteyl-tRNA[Ser]Sec-Sec-specific elongation factor (EFsec) complex in vitro and facilitates interaction between EFsec and SBP2 in vivo . The domains responsible for these interactions represent important structural determinants.

• Nuclear-cytoplasmic shuttling: TRNAU1AP appears to influence the subcellular localization of other selenoprotein synthesis factors. When co-expressed with SepSecS, TRNAU1AP induces SepSecS to localize to the cytoplasm rather than the nucleus . The structural elements that enable this trafficking function are under investigation.

• Post-translational modifications: The discrepancy between calculated (32 kDa) and observed (36 kDa and 21 kDa) molecular weights suggests potential post-translational modifications that may regulate TRNAU1AP function.