GOT2 Antibody

What is GOT2 and why is it a significant research target?

GOT2 is a mitochondrial transaminase that plays an essential role in the intracellular NAD(H) redox balance through its function in the malate-aspartate shuttle. Beyond this canonical role, GOT2 has been discovered to bind directly to fatty acid ligands that regulate the nuclear receptor PPARδ, impacting transcriptional activity and influencing the immune microenvironment in cancer contexts. GOT2's significance as a research target stems from its upregulation in various cancers (including breast and pancreatic cancer) and its involvement in suppressing antitumor immunity, making it a potential therapeutic target .

What applications are GOT2 antibodies most commonly used for?

GOT2 antibodies are validated for multiple research applications including:

• Western blot analysis (dilution ranges of 1:500-1:3000)

• Immunocytochemistry/Immunofluorescence (dilution ranges of 1:100-1:1000)

• Immunohistochemistry-Paraffin (dilution ranges of 1:100-1:1000)

• Flow cytometry (typically 0.40 μg per 10^6 cells in a 100 μl suspension)

Different applications may require specific antibody formulations and optimization of dilution ratios to achieve optimal results .

What is the expected cellular localization when using GOT2 antibodies?

While GOT2 is canonically described as a mitochondrial protein localized to the mitochondrial matrix, research has revealed that it can also be found in other cellular compartments. Importantly, a pool of GOT2 has been observed to localize to the nucleus in certain cancer cells, including murine premalignant lesions and human pancreatic ductal adenocarcinoma (PDAC). Additionally, some GOT2 localization to the cell membrane has been reported. When designing localization studies using GOT2 antibodies, researchers should consider these multiple potential localizations and use appropriate controls to distinguish between mitochondrial, nuclear, and other pools of the protein .

What species reactivity can be expected with commercial GOT2 antibodies?

Commercial GOT2 antibodies show reactivity across multiple species. For example:

• Human samples (primary validation for most antibodies)

• Mouse samples (commonly validated)

• Rat samples

• Porcine/pig samples (with approximately 89% sequence identity to human in some regions)

• Chicken samples (with approximately 82% sequence identity to human in some regions)

• Potential cross-reactivity with Xenopus laevis (approximately 81% sequence identity)

When using these antibodies in non-human models, it's important to verify reactivity through pilot experiments, as the degree of conservation in the immunogen region will affect antibody performance .

How can GOT2 antibodies be utilized to investigate its non-canonical functions in cancer?

To investigate GOT2's non-canonical roles in cancer, researchers can employ multiple advanced approaches:

• Nuclear vs. Mitochondrial Fraction Analysis: Use subcellular fractionation followed by western blotting with GOT2 antibodies to quantify the relative distribution between mitochondrial and nuclear pools. This is critical when investigating GOT2's role in PPARδ regulation, as nuclear translocation appears central to this function.

• Co-immunoprecipitation Studies: GOT2 antibodies can be used in co-IP experiments to pull down GOT2 and identify its interaction partners, particularly fatty acid ligands and transcriptional regulators like PPARδ.

• Chromatin Immunoprecipitation: To investigate potential direct or indirect interactions with DNA, ChIP assays using GOT2 antibodies can help determine if GOT2 associates with particular genomic regions.

• Proximity Ligation Assays: This technique can visualize and quantify in situ protein-protein interactions between GOT2 and suspected binding partners like PPARδ.

These approaches can help elucidate how GOT2 influences transcriptional activity beyond its established metabolic functions .

How should antibody selection differ when investigating GOT2's immune evasion mechanisms versus metabolic functions?

When investigating these distinct functions, antibody selection should consider:

For immune evasion mechanisms:

• Prioritize antibodies validated for in situ techniques (IHC, IF) to visualize spatial relationships between GOT2-expressing cancer cells and immune cell infiltrates

• Consider antibodies that can detect both mitochondrial and nuclear pools of GOT2

• Select antibodies compatible with multiplex immunostaining to simultaneously visualize GOT2, immune cell markers (CD4, CD8, CD11b), and functional markers (Ki-67, PD-1, GRZB)

For metabolic functions:

• Prioritize antibodies validated for enzymatic activity assays or that don't interfere with the active site

• Select antibodies that work effectively in mitochondrial fraction analysis

• Consider antibodies that work in native conditions to preserve protein-protein interactions within metabolic complexes

The research context dictates antibody selection strategy, as certain epitopes may be masked or exposed differently depending on GOT2's functional state and binding partners .

What considerations are important when using GOT2 antibodies in studies involving GOT2 genetic manipulation?

When using GOT2 antibodies in knockout, knockdown, or overexpression studies:

• Epitope Preservation: Verify that genetic manipulation techniques (CRISPR/Cas9, shRNA) don't alter the epitope recognized by the antibody. This is especially important when using domain-specific genetic modifications.

• Expression Level Validation: Use quantitative western blotting with GOT2 antibodies to confirm the degree of knockdown or overexpression. Research has shown that shRNA knockdown may result in partial recovery of GOT2 expression over time, necessitating temporal validation .

• Specificity Controls: Always include appropriate controls (wild-type vs. knockout samples) to confirm antibody specificity, particularly when using reconstitution experiments with modified GOT2 (e.g., NLS-GOT2 for nuclear targeting).

• Detection of Compensatory Mechanisms: GOT2 antibodies can help detect potential compensatory upregulation of related proteins (like GOT1) following GOT2 manipulation.

• Functional Domain Analysis: When using domain mutants (e.g., fatty acid binding site mutations), select antibodies that recognize regions distinct from the mutation sites to ensure detection capability is maintained .

What are the optimal fixation and antigen retrieval methods for GOT2 detection in tissue sections?

For optimal GOT2 detection in tissue sections:

The choice of fixation and antigen retrieval methods significantly impacts the detection of different GOT2 pools. Citrate buffer-based antigen retrieval has been validated for detection in mouse brain tissues. For applications requiring detection of nuclear GOT2, optimization of these parameters is particularly important .

How should researchers approach western blot analysis of GOT2 considering its molecular weight variations?

When performing western blot analysis of GOT2:

How can researchers address discrepancies between different GOT2 antibodies in experimental results?

When faced with discrepancies between different GOT2 antibodies:

• Compare Immunogen Information: Different antibodies may target distinct epitopes within GOT2. Some commercial antibodies target recombinant proteins encompassing "a sequence within the center region of human GOT2," while others might target N-terminal or C-terminal regions. These differences can affect detection capabilities, especially if certain epitopes are masked in protein complexes or modified states .

• Validation Using Genetic Controls: Always validate antibodies using appropriate genetic controls:

  • GOT2 knockout cell lines (generated via CRISPR/Cas9)

  • siRNA or shRNA knockdown samples (noting that partial recovery of expression may occur over time)

  • Overexpression systems with tagged versions for co-detection

• Cross-Validate with Multiple Techniques: If an antibody works in western blot but not in IHC, or vice versa, this may indicate epitope accessibility issues rather than antibody specificity problems.

• Application-Specific Optimization: Each application (WB, IHC, IF, flow cytometry) may require different antibody concentrations and conditions. Starting with manufacturer recommendations and then titrating is advisable .

What strategies can improve detection of GOT2's nuclear localization in cancer tissues?

To enhance detection of nuclear GOT2 in cancer tissues:

• Optimization of Nuclear Extraction: Use gentle extraction protocols that preserve nuclear membrane integrity while effectively isolating nuclear proteins.

• Selection of Nuclear-Detection Validated Antibodies: Some GOT2 antibodies may preferentially detect mitochondrial epitopes. Select antibodies validated for nuclear GOT2 detection.

• Co-staining Approaches: Implement dual immunofluorescence with established nuclear markers and mitochondrial markers to clearly distinguish the nuclear GOT2 pool.

• High-Resolution Imaging: Use confocal microscopy or super-resolution techniques to clearly distinguish between perinuclear mitochondrial staining and true nuclear localization.

• Nuclear Targeting Experiments: As demonstrated in research, comparing wild-type GOT2 with NLS-GOT2 (nuclear localization sequence-tagged GOT2) can help validate nuclear functions and optimize detection methods .

How should researchers interpret varying GOT2 expression patterns in immune-related cancer studies?

When interpreting GOT2 expression patterns in immune-related cancer studies:

How might GOT2 antibodies facilitate research into the GOT2-PPARδ interaction as a therapeutic target?

GOT2 antibodies could advance research into the GOT2-PPARδ interaction through:

• Structural Studies: Using antibodies recognizing different GOT2 domains to map the specific regions involved in PPARδ interaction, particularly the five putative fatty acid binding sites identified based on hydrophobicity .

• Dynamic Interaction Analysis: Employing antibodies in live-cell imaging techniques to track GOT2-PPARδ interactions in real-time, potentially revealing contextual regulation.

• Drug Discovery Applications: Developing screening assays using GOT2 antibodies to identify compounds that disrupt the GOT2-PPARδ interaction, potentially leading to novel immunotherapy adjuvants.

• Biomarker Development: Exploring whether antibodies against phosphorylated or otherwise modified forms of GOT2 could serve as biomarkers for predicting immunotherapy responsiveness in PDAC and other cancers .

• Combination Therapy Assessment: Using GOT2 antibodies to monitor changes in GOT2 expression, localization, and interaction partners during combination treatments targeting both metabolic and immune pathways.