APXT Antibody

What is the APTX protein and why is it important in research?

Aprataxin (APTX) is a ~39-41 kDa protein involved in DNA repair pathways. The protein is encoded by the APTX gene, which has been identified with the GenBank accession number BC104881 and NCBI GeneID 54840 . APTX is also known by several synonyms including AXA1 and FHA-HIT (Forkhead-associated domain histidine triad-like protein) . This protein plays a critical role in repairing DNA single-strand breaks, making it relevant to research on neurodegenerative disorders and DNA damage response mechanisms. Understanding APTX function requires reliable detection methods, which is why high-quality antibodies against this protein are essential tools in molecular and cellular biology research.

What applications are APTX antibodies primarily used for in research?

APTX antibodies are primarily utilized for Western blotting (WB), immunohistochemistry (IHC), and immunofluorescence/immunocytochemistry (IF/ICC) applications . These antibodies can also be used in enzyme-linked immunosorbent assays (ELISA) . For Western blot applications, researchers typically employ dilutions ranging from 1:500 to 1:2000, while immunohistochemistry and immunofluorescence applications generally require dilutions between 1:50 and 1:500 . The versatility of these antibodies allows researchers to detect APTX protein expression in various experimental contexts, from cell lysates to tissue sections, providing comprehensive insights into protein localization, expression levels, and potential interactions.

How should researchers validate APTX antibody specificity?

Validating antibody specificity is crucial for reliable experimental results. For APTX antibodies, researchers should first verify that the observed molecular weight matches the expected size of approximately 39-41 kDa . Cross-validation using multiple detection methods is recommended - for example, comparing Western blot results with immunofluorescence patterns. Researchers can also utilize positive control samples known to express APTX, such as HEK-293T cells, which have been documented to show clear APTX expression in Western blot analyses . Additionally, examining tissue-specific expression patterns can provide further validation - for instance, APTX antibodies have been successfully applied to human ovarian cancer and intrahepatic cholangiocarcinoma tissue sections .

What sample preparation protocols are recommended for APTX antibody applications?

For Western blotting, standard SDS-PAGE protocols are effective with APTX antibodies. Cells should be lysed in an appropriate buffer, proteins denatured, and samples loaded for electrophoretic separation . For immunohistochemistry applications using paraffin-embedded tissues, heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) has been shown to yield optimal results . When preparing samples for immunofluorescence, fixation with 4% paraformaldehyde (PFA) is recommended, as demonstrated in successful experiments with HepG2 cells . Across all applications, maintaining appropriate blocking steps and incubation times is essential - room temperature incubation for 1.5 hours has been documented to work well for Western blot applications .

What are the common troubleshooting issues with APTX antibody experiments?

Common issues researchers may encounter include weak or absent signals, non-specific binding, and background noise. For weak signals, optimizing antibody concentration, extending incubation times, or improving antigen retrieval methods may help. When experiencing high background, increasing the stringency of wash steps or using alternative blocking agents can improve results. Non-specific binding might be addressed by titrating the primary antibody concentration or using more specific detection methods. For immunohistochemistry applications specifically, optimizing antigen retrieval methods is crucial - while Tris-EDTA buffer (pH 9.0) is recommended, citrate buffer (pH 6.0) can serve as an alternative method .

How can researchers optimize APTX antibody dilutions for maximum signal-to-noise ratio?

Optimization of antibody dilutions is critical for achieving the best signal-to-noise ratio. For APTX antibodies, researchers should conduct titration experiments using a range of dilutions centered around the manufacturer's recommendations (1:500-1:2000 for WB, 1:50-1:500 for IHC and IF/ICC) . The optimal dilution will depend on the specific application, sample type, and detection system. For Western blot applications, researchers can prepare a standard curve using known quantities of recombinant APTX protein to determine the linear range of detection. For immunohistochemistry, comparing serial dilutions on identical tissue sections can identify the concentration that maximizes specific staining while minimizing background. When using fluorescence-based detection methods, researchers should also consider the autofluorescence properties of their samples and select appropriate controls and counterstains accordingly.

What advanced epitope mapping approaches can be used to characterize APTX antibodies?

Advanced epitope mapping can provide valuable insights into antibody specificity and potential cross-reactivity. Researchers can employ peptide arrays consisting of overlapping synthetic peptides spanning the entire APTX sequence to identify specific binding regions. Another approach involves creating domain-deletion mutants of APTX to determine which structural elements are required for antibody recognition. For more precise mapping, hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify specific amino acid residues involved in antibody binding. Understanding the exact epitope recognized by an APTX antibody can inform experimental design and interpretation, particularly when studying protein variants, isoforms, or species homologs. This approach parallels strategies used in other antibody research fields, where identifying immunodominant epitopes has improved diagnostic and predictive value .

How do post-translational modifications affect APTX antibody recognition?

Post-translational modifications (PTMs) of APTX, such as phosphorylation, ubiquitination, or SUMOylation, can significantly impact antibody recognition. These modifications may either mask epitopes or create new conformational states that alter antibody binding affinity. Researchers investigating PTMs should consider using phospho-specific or modification-specific antibodies in combination with general APTX antibodies. Alternatively, treating samples with phosphatases or deubiquitinating enzymes before antibody application can help determine if PTMs affect detection. When interpreting experimental results, researchers should consider that differences in signal intensity might reflect changes in protein modification states rather than expression levels. This consideration is particularly important when studying APTX in different cellular contexts or in response to various stimuli that might alter its post-translational modification profile.

How can APTX antibodies be utilized in protein-protein interaction studies?

APTX antibodies can be valuable tools for investigating protein-protein interactions through co-immunoprecipitation (co-IP), proximity ligation assays (PLA), or immunofluorescence co-localization studies. For co-IP experiments, researchers should optimize antibody amounts and binding conditions to efficiently capture APTX and its interacting partners without disrupting protein complexes. When using APTX antibodies for PLA, careful optimization of fixation methods and antibody compatibility with other primary antibodies is essential. Researchers can also employ APTX antibodies in pull-down assays followed by mass spectrometry to identify novel interaction partners. These approaches can provide valuable insights into APTX's role in various cellular processes and potential involvement in protein complexes that regulate DNA repair pathways.

What considerations are important when using APTX antibodies in chromatin immunoprecipitation (ChIP) experiments?

Although not explicitly mentioned in the search results, APTX's role in DNA repair suggests potential chromatin association, making ChIP a relevant application. When adapting APTX antibodies for ChIP experiments, researchers should first verify that the antibody can recognize native (non-denatured) APTX protein bound to DNA. Optimizing crosslinking conditions is crucial, as excessive crosslinking might mask epitopes while insufficient crosslinking could result in poor enrichment. Sonication parameters should be carefully adjusted to generate chromatin fragments of appropriate size (typically 200-500 bp). Including input controls, IgG controls, and positive controls (antibodies against known chromatin-associated proteins) is essential for proper data interpretation. Researchers should also consider the potential impact of fixation on epitope accessibility and may need to test different antibody concentrations than those used for other applications.

How can computational modeling enhance APTX antibody design and specificity?

Recent advances in computational modeling can significantly improve antibody design and specificity prediction. Biophysics-informed modeling approaches, similar to those used for other antibodies, can help design APTX antibodies with customized specificity profiles . These computational methods can identify different binding modes associated with particular ligands and predict antibody-antigen interactions with high accuracy. By combining experimental data with computational modeling, researchers can design antibodies that selectively recognize specific epitopes or protein conformations of APTX. This approach can be particularly valuable when developing antibodies that distinguish between closely related protein variants or specific post-translational modifications. The integration of high-throughput sequencing data with computational analysis represents a powerful strategy for expanding the repertoire of available APTX antibodies beyond those generated through traditional selection methods .