AARSD1 Antibody

What is AARSD1 and what role does it play in cellular function?

AARSD1 (Alanyl-tRNA synthetase domain-containing protein 1) is an enzyme that functions in trans to edit the amino acid moiety from incorrectly charged tRNA(Ala) . It belongs to the class-II aminoacyl-tRNA synthetase family and the Alax-L subfamily. The protein plays a crucial role in protein synthesis by ensuring translational fidelity through its editing capabilities. AARSD1 has been localized to both the cytoplasm and nucleus, suggesting multiple cellular functions . Beyond its canonical role in tRNA editing, recent research suggests aminoacyl-tRNA synthetases like AARSD1 may have noncanonical functions related to various disease processes, including autoimmune diseases, cancer, and neurological disorders .

What experimental applications are validated for AARSD1 antibodies?

AARSD1 antibodies, such as the PACO40482, have been validated for multiple experimental applications including Western blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), and Immunohistochemistry (IHC) . These validations ensure reliable detection of the target protein across different experimental conditions. For Western blot applications, the antibody has been successfully tested on various cell lysates including HeLa and U251 cell lines, demonstrating specific detection of the AARSD1 protein at the expected molecular weight of approximately 46 kDa . In immunohistochemistry applications, the antibody has shown effective staining in paraffin-embedded human tissues including testis tissue and lung cancer samples .

What are the recommended experimental conditions for using AARSD1 antibodies?

The optimal working conditions for AARSD1 antibodies vary depending on the specific application. Based on validated protocols, the following dilutions are recommended:

For Western blot applications, researchers should expect to observe bands at approximately 46 kDa, although predicted band sizes may include 59 and 66 kDa depending on the isoform detected . For optimal results, use goat polyclonal anti-rabbit IgG at 1/10000 dilution as a secondary antibody .

How can researchers validate the specificity of their AARSD1 antibody results?

Validating antibody specificity is critical for ensuring experimental reliability. A comprehensive validation approach should include:

• Positive and negative controls: Include cell lines known to express AARSD1 (like HeLa) alongside those with low or no expression as controls .

• Multiple detection methods: Confirm results using orthogonal techniques such as combining Western blot with immunohistochemistry or immunofluorescence .

• Peptide competition assays: Pre-incubate the antibody with the immunogen peptide to demonstrate signal reduction in the presence of the blocking peptide.

• Immunoprecipitation followed by mass spectrometry (IP-MS): This technique can validate that the antibody captures the endogenous protein from mammalian cell lysates. High-quality antibodies should pull down the target protein with minimal non-specific binding .

• Cross-reactivity testing: Test the antibody against multiple related proteins to ensure it does not cross-react with other aminoacyl-tRNA synthetases. Suspension bead assays like Luminex can be used to test antibodies against multiple related antigens simultaneously .

How does AARSD1 function in the multi-tRNA synthetase complex and what are the experimental implications?

AARSD1 operates within the broader context of aminoacyl-tRNA synthetases, some of which form multi-tRNA synthetase complexes. When investigating AARSD1:

• Complex co-immunoprecipitation: When targeting individual members of multi-tRNA synthetase complexes with antibodies, researchers can detect all members of the complex co-immunoprecipitating with the target in various cell types . This provides insight into protein-protein interactions.

• Functional overlap consideration: Experiments should account for potential functional redundancy or compensation by other aminoacyl-tRNA synthetases in knockdown or knockout studies.

• Domain-specific targeting: Since AARSD1 contains multiple functional domains, researchers may need to design experiments that target specific domains for comprehensive functional analysis .

• Noncanonical function analysis: Beyond its canonical role in protein synthesis, experimental designs should consider potential noncanonical functions when interpreting results .

What are the considerations for using AARSD1 antibodies in disease-related research?

Recent findings have linked aminoacyl-tRNA synthetases like AARSD1 to various pathological conditions:

• Cancer research applications: AARSD1 antibodies have been validated for use in lung cancer tissue by immunohistochemistry . Researchers should optimize staining protocols for specific cancer types and consider quantitative analysis of expression levels.

• Neurodegenerative disorders: When studying potential links between AARSD1 dysregulation and neurological conditions, researchers should consider co-staining with neuronal markers and employ specialized tissue preparation techniques.

• Translational fidelity mechanisms: Since AARSD1 functions in editing incorrectly charged tRNAs, experimental designs investigating translational errors should include AARSD1 activity assays alongside expression analysis .

• Therapeutic target investigation: When evaluating AARSD1 as a potential therapeutic target, consider both its canonical and noncanonical functions to anticipate potential off-target effects .

How do recombinant antibodies for AARSD1 compare with traditional polyclonal and monoclonal antibodies?

Recombinant antibodies offer distinct advantages for AARSD1 research:

• Reproducibility: Unlike traditional polyclonal antibodies that can vary between lots, recombinant antibodies provide consistent performance across batches .

• Specificity engineering: Recombinant technologies allow for the selection of highly specific binding regions, potentially reducing cross-reactivity with related aminoacyl-tRNA synthetases .

• Validation methodology: Recombinant antibodies can undergo more stringent validation processes, including phage display selections against multiple related antigens to ensure specificity .

• Sequence transparency: The sequences of recombinant antibodies are typically deposited in public databases, enabling better reproducibility and potential modification for specialized applications .

• Format flexibility: Recombinant antibodies can be produced in various formats (scFv, Fab, full IgG) to optimize performance for specific applications like immunofluorescence or pull-down assays .

What are common troubleshooting strategies for AARSD1 antibody applications?

When encountering issues with AARSD1 antibody applications, consider the following troubleshooting approaches:

• Western blot optimization:

  • If detecting multiple bands, optimize primary antibody concentration (recommended 0.8μg/ml)

  • Verify sample preparation method preserves protein integrity

  • Include positive controls (HeLa or U251 cell lysates)

  • Adjust blocking conditions to reduce background

• Immunohistochemistry troubleshooting:

  • Optimize antigen retrieval methods for different tissue types

  • Test multiple dilutions around the recommended 1:100 dilution

  • Extend primary antibody incubation time for weak signals

  • Use amplification systems for low-abundance targets

• Signal specificity verification:

  • Perform peptide competition assays

  • Include knockout/knockdown controls when possible

  • Compare results with alternative AARSD1 antibodies (such as PACO00350 or PACO07514)

How can researchers optimize immunoprecipitation experiments with AARSD1 antibodies?

For successful immunoprecipitation of AARSD1:

• Lysis buffer optimization: Use buffers that maintain protein-protein interactions if studying complex formation, or more stringent buffers if studying only AARSD1.

• Pre-clearing strategy: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody amount titration: Determine the optimal antibody-to-lysate ratio through titration experiments.

• Co-IP considerations: When studying multi-tRNA synthetase complexes, gentler lysis and washing conditions may be required to maintain complex integrity .

• Validation by mass spectrometry: Confirm the identity of immunoprecipitated proteins through mass spectrometry, which can also reveal potential interaction partners .

How can AARSD1 antibodies contribute to understanding translational quality control mechanisms?

AARSD1's role in editing incorrectly charged tRNAs positions it as a key player in translational quality control. Researchers can:

What considerations should be made when using AARSD1 antibodies for studying its noncanonical functions?

While AARSD1's canonical role involves tRNA editing, investigating its potential noncanonical functions requires specific considerations:

• Subcellular localization studies: Use immunofluorescence with AARSD1 antibodies to track its localization under different cellular conditions, potentially revealing non-canonical functions in specific compartments .

• Protein-protein interaction networks: Employ co-immunoprecipitation followed by mass spectrometry to identify novel interaction partners beyond the tRNA synthesis machinery .

• Non-enzymatic function investigation: Design experiments that can distinguish between AARSD1's enzymatic activity and potential structural or signaling roles.

• Disease-context studies: Examine AARSD1 expression and localization patterns in disease contexts where other aminoacyl-tRNA synthetases have shown noncanonical functions .