SDHAF2 Antibody

What is SDHAF2 and why is it important in research?

SDHAF2 (also known as PGL2 or SDH5) is a mitochondrial protein essential for the covalent attachment of the FAD cofactor to the SDHA subunit of succinate dehydrogenase (Complex II). This protein is crucial for electron transport chain functionality and cellular respiration. Research interest in SDHAF2 has intensified due to its tumor suppressor role, with mutations linked to hereditary head and neck paragangliomas (HNPGL) and other neuroendocrine tumors. Understanding SDHAF2 function provides insights into mitochondrial biology, cancer development, and potential therapeutic targets for complex II deficiencies .

What types of SDHAF2 antibodies are available for research?

Researchers can utilize several types of SDHAF2 antibodies, including rabbit polyclonal antibodies that recognize human SDHAF2 with high specificity and chicken polyclonal IgY antibodies that exhibit cross-reactivity with mouse and rat SDHAF2 . These antibodies are generated using synthetic peptides from specific regions of SDHAF2, typically from the amino terminus. Validation testing confirms their reactivity in multiple applications, including Western blot, ELISA, and immunohistochemistry. The choice between these antibody types depends on the experimental design, target species, and specific application requirements .

What is the molecular weight and structure of SDHAF2 protein?

SDHAF2 has a calculated molecular weight of approximately 19.6 kDa. Structurally, it contains a conserved five-helix bundle that interacts with SDHA. Human SDHAF2 is approximately 50% larger than its bacterial homologs (like SdhE), with additional N-terminal and C-terminal extensions that are unique to mitochondrial homologs. These extensions contribute to the stability of the SDHA-SDHAF2 complex by increasing the buried surface area to approximately 1,922 Ų, compared to 1,072-1,458 Ų in bacterial homologs. The protein contains intrinsically disordered regions at its termini that become ordered upon binding to SDHA, as demonstrated by NMR spectroscopy studies .

What applications are SDHAF2 antibodies validated for?

SDHAF2 antibodies have been validated for multiple research applications. Commercial antibodies are typically guaranteed for Western blot (WB) and ELISA applications, with some also validated for immunohistochemistry (IHC) and immunocytochemistry with immunofluorescence (ICC-IF). When using these antibodies in Western blot applications, they are typically applied at concentrations around 1 μg/mL. For optimal results in any application, researchers should perform titration experiments to determine the ideal antibody concentration for their specific experimental conditions and sample types .

How should researchers optimize Western blot protocols for SDHAF2 detection?

For optimal Western blot detection of SDHAF2, researchers should consider the following methodological approach: (1) Prepare tissue or cell lysates with protease inhibitors to prevent degradation of the target protein; (2) Separate proteins using 12-15% SDS-PAGE gels suitable for low molecular weight proteins (~20 kDa); (3) Use PVDF membranes for transfer with standard transfer buffer; (4) Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour; (5) Incubate with primary SDHAF2 antibody at approximately 1 μg/mL concentration overnight at 4°C; (6) Use appropriate HRP-conjugated secondary antibodies (anti-rabbit or anti-chicken depending on the primary antibody host); (7) Include positive controls such as liver tissue lysate; and (8) To confirm specificity, perform a blocking peptide experiment where the antibody is pre-incubated with the immunizing peptide before application to the membrane, which should abolish the specific signal .

What are the recommended storage conditions for SDHAF2 antibodies?

SDHAF2 antibodies require specific storage conditions to maintain their functionality and specificity. These antibodies can typically be stored at 4°C for up to three months for ongoing experiments. For long-term storage, maintaining them at -20°C is recommended, where they remain stable for up to one year. It is crucial to avoid repeated freeze-thaw cycles as these can cause antibody degradation and reduced performance. Antibodies should never be exposed to prolonged high temperatures. Most SDHAF2 antibodies are supplied in PBS containing 0.02% sodium azide as a preservative, which helps maintain stability. Proper aliquoting upon receipt can minimize freeze-thaw cycles when using the antibody for multiple experiments over time .

How can SDHAF2 antibodies be used to study complex II assembly mechanisms?

To investigate Complex II assembly mechanisms, researchers can employ SDHAF2 antibodies in co-immunoprecipitation experiments to isolate SDHAF2-SDHA complexes and identify additional interaction partners or post-translational modifications. Combine this with sucrose gradient centrifugation to separate assembly intermediates of different molecular weights, followed by Western blot analysis with SDHAF2 and SDHA antibodies to track assembly progression. For in-depth structural analysis, use crosslinking mass spectrometry with SDHAF2 antibodies for immunopurification to capture transient interaction states. Additionally, implement pulse-chase experiments with metabolic labeling followed by immunoprecipitation to track the kinetics of FAD incorporation and complex assembly. Complementary approaches include proximity ligation assays to visualize SDHAF2-SDHA interactions in situ and CRISPR-mediated tagging of SDHAF2 combined with live-cell imaging to monitor the spatiotemporal dynamics of complex II assembly .

What is the role of SDHAF2 in FAD cofactor attachment, and how can this be studied?

SDHAF2 facilitates the covalent attachment of FAD to the SDHA subunit, specifically to His99 in human SDHA. This flavinylation process is essential for complex II activity and is enhanced by small molecule dicarboxylates like oxaloacetate. To study this mechanism, researchers can use a multi-faceted approach: (1) Implement in vitro flavinylation assays with purified components (SDHA, SDHAF2, FAD) and monitor covalent attachment using fluorescence detection of FAD; (2) Use site-directed mutagenesis to modify key residues like SDHAF2 G78 (which forms a hydrogen bond with SDHA H99) and assess the impact on flavinylation efficiency; (3) Conduct comparative studies with disease-associated mutants to understand the molecular basis of pathogenicity; (4) Employ structural techniques like X-ray crystallography or cryo-EM to visualize the SDHA-SDHAF2-dicarboxylate complex at different stages of flavinylation; and (5) Develop FRET-based assays to monitor the conformational changes associated with FAD attachment in real-time .

How are SDHAF2 antibodies used in studying paraganglioma and related disorders?

In paraganglioma research, SDHAF2 antibodies serve multiple crucial functions. They enable immunohistochemical screening of tumor tissue to assess SDHAF2 expression patterns and localization, which can help distinguish between sporadic and hereditary paragangliomas. Researchers can perform comparative protein expression analyses between normal tissue, paragangliomas with SDHAF2 mutations, and those with mutations in other SDH subunits to identify specific molecular signatures. The antibodies also facilitate the identification of novel SDHAF2 interaction partners in paraganglioma tissues through co-immunoprecipitation followed by mass spectrometry. Additionally, they can be used in chromatin immunoprecipitation sequencing (ChIP-seq) experiments to investigate potential nuclear roles of SDHAF2 in gene regulation. For clinical applications, SDHAF2 antibodies enable the development of immunohistochemical screening algorithms to identify patients who should undergo genetic testing for SDHAF2 mutations, particularly in cases of hereditary head and neck paragangliomas with maternal imprinting patterns .

How can researchers distinguish between false positives and true signals when using SDHAF2 antibodies?

To distinguish between false positives and true SDHAF2 signals, researchers should implement a comprehensive validation strategy: (1) Always include blocking peptide controls by pre-incubating the antibody with the immunizing peptide, which should eliminate specific signals; (2) Include positive control samples with known SDHAF2 expression (such as liver tissue) and negative controls like cell lines with SDHAF2 knockdown; (3) Verify signal specificity by comparing results from multiple antibodies targeting different epitopes of SDHAF2; (4) Confirm the appropriate molecular weight (~19.6 kDa) in Western blot applications; (5) For immunohistochemistry, validate the expected mitochondrial localization pattern through co-staining with established mitochondrial markers; (6) Use recombinant SDHAF2 protein as a standard for quantification and specificity assessment; and (7) Confirm critical findings with orthogonal techniques such as mass spectrometry to validate antibody-based results .

How should researchers interpret SDHAF2 expression patterns in different tissues and disease states?

When interpreting SDHAF2 expression patterns across tissues and disease states, researchers should consider several factors. First, baseline SDHAF2 expression varies naturally between tissues, with higher expression typically observed in metabolically active organs like liver and heart. Expression patterns should be analyzed in context with other Complex II components (SDHA, SDHB, SDHC, SDHD) to identify potential compensatory mechanisms or assembly defects. In disease states, particularly paragangliomas, reduced SDHAF2 expression might indicate loss-of-function mutations, while abnormal subcellular localization could suggest impaired protein trafficking. Researchers should also assess post-translational modifications of SDHAF2 using phospho-specific or ubiquitin-specific antibodies to identify regulatory mechanisms. For comprehensive interpretation, integrate antibody-based expression data with functional assays of Complex II activity, FAD incorporation efficiency, and mitochondrial respiration measurements. Additionally, correlate SDHAF2 expression patterns with clinical parameters and genetic testing results when studying hereditary paraganglioma syndromes .

What are the common pitfalls in experimental design when using SDHAF2 antibodies?

Common experimental design pitfalls when using SDHAF2 antibodies include: (1) Inadequate antibody validation - always confirm specificity through blocking peptide experiments and appropriate controls; (2) Overlooking potential cross-reactivity with other SDHAF family members despite manufacturer predictions of specificity; (3) Insufficient optimization of antibody concentration for specific applications and tissue types; (4) Improper sample preparation that affects epitope accessibility, particularly for mitochondrial proteins that may require special extraction protocols; (5) Failure to account for maternal imprinting effects when studying SDHAF2 in hereditary paraganglioma cases, which can lead to misleading genotype-phenotype correlations; (6) Neglecting to verify subcellular localization of SDHAF2 alongside expression levels; (7) Using inappropriate normalization controls that don't account for mitochondrial mass variations between samples; and (8) Overlooking the impact of specific fixation methods on epitope preservation in immunohistochemistry applications. To mitigate these issues, researchers should conduct preliminary optimization experiments and include comprehensive controls tailored to their specific research questions .

How can SDHAF2 antibodies contribute to understanding the role of CII low in metabolic regulation?

SDHAF2 antibodies can provide crucial insights into the emerging concept of CII low - an alternative low molecular weight form of Complex II that includes SDHA, SDHAF2, and SDHAF4. This complex has been proposed as a storage form during bioenergetic stress that regulates metabolic balance. To investigate this phenomenon, researchers can employ SDHAF2 antibodies in several sophisticated approaches: (1) Use differential centrifugation combined with immunoprecipitation to isolate and characterize CII low complexes from mitochondria under various metabolic conditions; (2) Implement blue native PAGE followed by Western blotting with SDHAF2 antibodies to separate and identify different Complex II assembly states; (3) Combine SDHAF2 immunoprecipitation with mass spectrometry to comprehensively identify all components and post-translational modifications of CII low; (4) Apply metabolic flux analysis in conjunction with SDHAF2 manipulation to determine how CII low levels affect TCA cycle activity and oxidative phosphorylation; and (5) Develop SDHAF2-based biosensors to monitor real-time changes in CII low formation in response to metabolic perturbations. These approaches could reveal how SDHAF2 contributes to mitochondrial signaling cascades important for bioenergetic balance beyond its known role in complex assembly .

What approaches can be used to study the relationship between SDHAF2 mutations and clinical phenotypes?

To investigate the relationship between SDHAF2 mutations and clinical phenotypes, researchers can employ a multi-dimensional approach: (1) Develop immunohistochemical protocols using SDHAF2 antibodies to screen paraganglioma tissue microarrays, correlating expression patterns with genetic testing and clinical outcomes; (2) Create isogenic cell lines with CRISPR-Cas9 to introduce specific SDHAF2 mutations (like G78R) and assess their functional consequences using antibody-based assays for protein-protein interactions and complex assembly; (3) Implement proximity-dependent biotin identification (BioID) with wild-type and mutant SDHAF2 to map changes in the interactome associated with pathogenic variants; (4) Conduct patient-derived organoid studies with SDHAF2 antibody staining to observe mutation effects in three-dimensional tissue contexts; (5) Utilize high-content imaging with SDHAF2 and metabolic stress markers to identify cellular phenotypes associated with different mutations; and (6) Develop mouse models with conditional SDHAF2 mutations combined with tissue-specific immunohistochemical analysis to understand organ-specific manifestations of SDHAF2 dysfunction. This comprehensive approach can help explain the variable disease presentation in patients with SDHAF2 mutations and potentially identify novel therapeutic targets .

How can researchers leverage SDHAF2 antibodies to investigate potential nuclear roles beyond mitochondrial function?

While SDHAF2 is primarily considered a mitochondrial protein, emerging evidence suggests potential extra-mitochondrial functions. To investigate possible nuclear roles, researchers can employ several sophisticated approaches using SDHAF2 antibodies: (1) Perform subcellular fractionation followed by Western blotting to detect SDHAF2 in nuclear extracts, with rigorous controls to exclude mitochondrial contamination; (2) Use chromatin immunoprecipitation sequencing (ChIP-seq) with SDHAF2 antibodies to identify potential DNA binding sites or association with chromatin-modifying complexes; (3) Implement proximity ligation assays to visualize potential interactions between SDHAF2 and nuclear proteins in situ; (4) Conduct mass spectrometry analysis of nuclear SDHAF2 immunoprecipitates to identify nuclear-specific interaction partners; (5) Develop cell lines with differentially tagged SDHAF2 variants targeted to either mitochondria or nucleus, followed by immunoprecipitation studies to compare distinct functions; and (6) Perform RNA immunoprecipitation to investigate potential roles in RNA processing or regulation. These approaches could reveal novel functions of SDHAF2 in gene expression regulation, DNA damage response, or nuclear-mitochondrial communication pathways that extend beyond its established role in Complex II assembly .

What quality control measures should be implemented when validating a new batch of SDHAF2 antibody?

When validating a new batch of SDHAF2 antibody, researchers should implement a comprehensive quality control protocol: (1) Perform Western blot analysis using positive control samples (such as liver tissue lysate) to confirm the expected molecular weight band at approximately 19.6 kDa; (2) Include negative controls like SDHAF2 knockout or knockdown samples to verify specificity; (3) Conduct a blocking peptide experiment by pre-incubating the antibody with its immunizing peptide to confirm signal abolishment; (4) Assess lot-to-lot consistency by comparing the new batch with previously validated batches using identical experimental conditions; (5) Verify species cross-reactivity if the antibody is claimed to recognize multiple species (human, mouse, rat); (6) Perform immunoprecipitation followed by mass spectrometry to confirm that SDHAF2 is indeed the primary pulled-down protein; (7) Test the antibody in all intended applications (Western blot, ELISA, IHC) to ensure consistent performance across platforms; and (8) Document all validation data, including images, protocols, and lot numbers, for future reference and reproducibility. This rigorous validation approach ensures reliable and consistent results when using SDHAF2 antibodies in critical research applications .

How can researchers design multiplexed immunofluorescence panels including SDHAF2?

To design effective multiplexed immunofluorescence panels including SDHAF2, researchers should follow this methodological approach: (1) Begin by selecting antibodies raised in different host species (e.g., rabbit anti-SDHAF2 and mouse anti-mitochondrial markers) to enable simultaneous detection with species-specific secondary antibodies; (2) Consider antibody isotypes carefully when using multiple primary antibodies from the same species to enable isotype-specific detection; (3) Optimize the signal-to-noise ratio for each antibody individually before combining them, determining the optimal concentration, incubation time, and antigen retrieval method for the SDHAF2 antibody; (4) Select fluorophores with minimal spectral overlap for secondary antibodies or directly conjugated primaries; (5) Include appropriate controls for each antibody in the panel, including single-stain controls to assess bleed-through and secondary-only controls to detect non-specific binding; (6) When studying SDHAF2 in relationship to mitochondrial function, include markers for mitochondrial subcompartments (matrix, inner membrane, outer membrane) to precisely localize SDHAF2; (7) Consider sequential detection protocols if cross-reactivity issues arise; and (8) Implement computational analysis workflows that can accurately segment and quantify colocalization patterns in cellular subcompartments .

How can researchers integrate SDHAF2 antibody data with omics approaches for comprehensive mitochondrial studies?

Integrating SDHAF2 antibody data with omics approaches enables comprehensive mitochondrial research through several methodological strategies: (1) Combine immunoprecipitation using SDHAF2 antibodies with mass spectrometry (IP-MS) to identify the complete interactome of SDHAF2 under different physiological conditions; (2) Correlate SDHAF2 protein levels detected by antibodies with transcriptomic data to identify potential post-transcriptional regulatory mechanisms; (3) Integrate immunohistochemistry or immunofluorescence spatial data with single-cell RNA sequencing to understand cell-type specific variations in SDHAF2 function; (4) Use SDHAF2 antibodies for Cleavable Cross-linker Immunoprecipitation (CLIP) experiments followed by proteomics to capture transient interactions during complex II assembly; (5) Implement ChIP-seq using SDHAF2 antibodies to identify potential moonlighting functions in transcriptional regulation; (6) Correlate SDHAF2 protein localization with metabolomic profiles to understand the metabolic consequences of SDHAF2 dysfunction; and (7) Develop computational workflows that integrate antibody-based quantification data with multi-omics datasets to construct comprehensive models of mitochondrial function in health and disease states .

What are the emerging applications of SDHAF2 antibodies in cancer metabolism research?

In cancer metabolism research, SDHAF2 antibodies are enabling several cutting-edge applications: (1) Tissue microarray analysis of diverse tumor types to establish SDHAF2 expression patterns as potential biomarkers or therapeutic targets; (2) Investigation of metabolic rewiring mechanisms in SDH-deficient tumors by combining SDHAF2 immunoprecipitation with metabolite profiling; (3) Development of companion diagnostic approaches using SDHAF2 immunohistochemistry to identify patients with dysfunctional complex II assembly who might benefit from specific metabolic therapies; (4) High-throughput screening of compounds that modulate SDHAF2-SDHA interactions, using antibodies in automated immunofluorescence or ELISA-based detection systems; (5) Monitoring changes in SDHAF2 expression and localization during hypoxia and other metabolic stresses characteristic of tumor microenvironments; (6) Investigating the relationship between SDHAF2 and oncometabolite accumulation (such as succinate) through co-localization studies with metabolite sensors; and (7) Exploring potential roles of SDHAF2 in regulating mitochondrial dynamics and mitophagy in cancer cells through live-cell imaging combined with fixed-cell antibody staining. These applications are revealing how alterations in complex II assembly contribute to the metabolic plasticity that enables cancer progression and therapy resistance .