SDHAF2 Antibody, FITC conjugated

What is SDHAF2 and why is it important in cellular biology?

SDHAF2 (also known as SDH5, PGL2) is a tumor suppressor gene encoding a protein required for the flavination of succinate dehydrogenase subunit SDHA. It plays an essential role in the assembly of the succinate dehydrogenase (SDH) complex, which is a critical component of both the tricarboxylic acid cycle and the mitochondrial electron transport chain. This complex couples the oxidation of succinate to fumarate with the reduction of ubiquinone to ubiquinol . The protein's importance lies in its role in maintaining proper mitochondrial function and energy metabolism, with disruptions potentially leading to paraganglioma development.

What are the key applications of SDHAF2 antibodies in research?

SDHAF2 antibodies are utilized across multiple research techniques including Western Blotting (WB), ELISA (EL), Immunohistochemistry (IHC), and Immunofluorescence (IF/ICC) . These applications enable researchers to investigate protein expression patterns, localization within tissues or cells, and protein-protein interactions involving SDHAF2. The antibodies are particularly valuable in studying mitochondrial dysfunction, paraganglioma development, and the functional consequences of SDHAF2 mutations in cellular models and patient samples.

What criteria should researchers consider when selecting a SDHAF2 antibody for specific applications?

When selecting a SDHAF2 antibody, researchers should evaluate: (1) Host species and clonality (rabbit polyclonal antibodies being common for SDHAF2) ; (2) Validated applications matching experimental needs (WB, IHC, IF, ELISA); (3) Species reactivity (human, mouse, rat) relevant to experimental models ; (4) Published validation data demonstrating specificity (citations and literature support); (5) Epitope location and whether it overlaps with known functional domains or mutation sites in SDHAF2; and (6) For FITC-conjugated antibodies specifically, fluorophore:protein ratio and signal stability should be considered to ensure optimal detection sensitivity.

How can researchers validate the specificity of SDHAF2 antibodies?

Validation of SDHAF2 antibodies should include multiple approaches: (1) Positive controls using tissues/cells known to express SDHAF2 (particularly mitochondria-rich tissues); (2) Negative controls using SDHAF2 knockout or knockdown models; (3) Peptide competition assays to confirm epitope specificity; (4) Western blot analysis confirming a single band at the expected molecular weight (approximately 20 kDa for human SDHAF2); (5) Comparison with alternative antibodies targeting different epitopes of SDHAF2; and (6) Subcellular localization studies confirming mitochondrial distribution pattern consistent with SDHAF2's known function .

What are the available reactivity profiles for SDHAF2 antibodies?

SDHAF2 antibodies are available with varying species reactivity profiles. Primary reactivity options include: (1) Human-specific antibodies, crucial for clinical sample analysis and human cell line research; (2) Mouse-reactive antibodies for murine model studies; (3) Rat-reactive antibodies for rat model systems; and (4) Multi-species reactive antibodies detecting conserved epitopes across human, mouse, and rat SDHAF2 . Researchers should select antibodies with validated reactivity for their experimental system, as cross-reactivity might not be guaranteed due to sequence differences in SDHAF2 across species.

What is the optimal protocol for immunofluorescence using FITC-conjugated SDHAF2 antibodies?

For optimal immunofluorescence with FITC-conjugated SDHAF2 antibodies:

• Fix cells with 4% paraformaldehyde (10 minutes at room temperature)

• Permeabilize with 0.2% Triton X-100 in PBS (5 minutes)

• Block with 5% normal serum in PBS (1 hour)

• Incubate with FITC-conjugated SDHAF2 antibody (1:50-1:200 dilution, overnight at 4°C in darkness)

• Wash 3× with PBS

• Counterstain with DAPI for nuclear visualization

• Mount with anti-fade mounting medium

Critical parameters include maintaining darkness during and after antibody incubation to prevent photobleaching, optimizing antibody concentration through titration experiments, and including a mitochondrial co-stain (such as MitoTracker) to confirm proper subcellular localization .

How should researchers design experiments to detect SDHAF2 in paraganglioma tissues?

For detecting SDHAF2 in paraganglioma tissues:

• Process tissue sections using standard formalin-fixed paraffin-embedded (FFPE) protocols

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0)

• Block endogenous peroxidase and biotin

• Apply validated SDHAF2 antibody (ideally one with confirmed IHC application)

• Compare staining patterns between tumor tissue and adjacent normal tissues

• Include positive controls (normal tissues with high SDHAF2 expression)

• Consider double-staining with markers for chief cells and sustentacular cells

Given that SDHAF2 mutations are associated with hereditary head and neck paragangliomas (HNPGL), researchers should pay particular attention to staining patterns in carotid body and vagal paragangliomas, as these represent 71% and 17% of tumors in SDHAF2 mutation carriers, respectively .

What controls are essential when performing western blotting for SDHAF2?

Essential controls for SDHAF2 western blotting include:

• Positive control: Lysate from cells/tissues known to express SDHAF2

• Negative control: Lysate from SDHAF2 knockdown/knockout cells

• Loading control: Detection of a housekeeping protein (β-actin, GAPDH)

• Molecular weight marker: To confirm band at expected size (~20 kDa)

• Peptide competition control: Pre-incubation of antibody with immunizing peptide

• Technical replicates: Multiple sample runs to confirm reproducibility

Additionally, researchers should optimize protein extraction protocols to ensure efficient recovery of mitochondrial proteins, considering that SDHAF2 is localized to mitochondria. Mitochondrial isolation may be necessary for detecting low-abundance SDHAF2 in certain cell types .

How can SDHAF2 antibodies be utilized to study the role of SDHAF2 in flavination of SDHA?

To study SDHAF2's role in SDHA flavination:

• Immunoprecipitation using SDHAF2 antibodies to pull down protein complexes

• Co-immunoprecipitation with SDHA followed by western blotting for SDHAF2

• Proximity ligation assays (PLA) to detect SDHAF2-SDHA interactions in situ

• Immunofluorescence co-localization of SDHAF2 and SDHA

• Biochemical assays measuring FAD incorporation into SDHA in the presence/absence of SDHAF2

• Comparative analysis of SDHAF2 and SDHA in normal versus mutant cells

This experimental approach enables investigation of the molecular mechanisms by which SDHAF2 facilitates the covalent attachment of FAD to SDHA, a critical process for succinate dehydrogenase complex assembly and function .

What methodologies can detect differences in SDHAF2 expression between normal and tumor tissues?

For detecting differential SDHAF2 expression between normal and tumor tissues:

• Quantitative immunohistochemistry with digital image analysis

  • Signal intensity quantification

  • Subcellular distribution analysis

  • Comparative scoring systems

• Tissue microarray (TMA) analysis

  • Multiple patient samples analyzed simultaneously

  • Paired normal-tumor tissue comparison

• Multiplexed immunofluorescence

  • Co-staining with cell type-specific markers

  • Analysis of SDHAF2 in specific cellular contexts

• Laser capture microdissection followed by western blotting

  • Isolation of specific cell populations

  • Direct quantification of protein levels

These approaches should be complemented with mRNA expression analysis to determine whether changes occur at transcriptional or post-transcriptional levels .

How can researchers investigate the impact of SDHAF2 mutations on protein-protein interactions within the SDH complex?

To investigate how SDHAF2 mutations affect protein-protein interactions:

• Generate cell models expressing wild-type and mutant SDHAF2 (particularly the c.232G>A mutation associated with paragangliomas)

• Perform co-immunoprecipitation assays with antibodies against:

  • SDHAF2 (to pull down interaction partners)

  • SDHA, SDHB, SDHC, SDHD (to assess complex formation)

• Conduct FRET or BRET analysis to measure proximity between fluorescently tagged SDHAF2 and other SDH components

• Use bimolecular fluorescence complementation (BiFC) to visualize interactions in living cells

• Employ quantitative mass spectrometry to identify differences in the interactome between wild-type and mutant SDHAF2

• Correlate interaction changes with SDH enzymatic activity and FAD incorporation

This comprehensive approach provides insights into the structural and functional consequences of SDHAF2 mutations on succinate dehydrogenase complex assembly .

How can FITC-conjugated SDHAF2 antibodies be used in the study of hereditary paraganglioma syndromes?

FITC-conjugated SDHAF2 antibodies can be applied in hereditary paraganglioma research through:

• Immunofluorescence analysis of tumor samples from patients with confirmed SDHAF2 mutations (particularly focusing on the PGL2 syndrome)

• Comparison of SDHAF2 protein expression and localization in tumor samples from patients with different SDH subunit mutations (SDHD, SDHC, SDHB vs. SDHAF2)

• Correlation of SDHAF2 staining patterns with clinical features including:

  • Age at onset (average 33 years in SDHAF2 mutation carriers)

  • Tumor multifocality (91% in SDHAF2 mutation carriers)

  • Tumor location (predominantly carotid and vagal)

• Fluorescence-activated cell sorting (FACS) of dissociated tumor cells based on SDHAF2-FITC signal

• Analysis of SDHAF2 expression in asymptomatic mutation carriers to identify early molecular changes

These approaches can provide insights into pathogenic mechanisms and potentially identify early biomarkers in individuals with SDHAF2 mutations .

What are the methodological considerations when using SDHAF2 antibodies to study maternal imprinting effects?

When studying maternal imprinting effects associated with SDHAF2:

• Sample collection:

  • Obtain samples from complete family pedigrees

  • Include individuals with maternal and paternal inheritance patterns

  • Collect samples from "risk-free carriers" (those inheriting mutations maternally)

• Expression analysis:

  • Compare SDHAF2 protein levels in individuals with paternally vs. maternally inherited mutations

  • Perform allele-specific expression analysis using tagged antibodies

• Epigenetic profiling:

  • Correlate SDHAF2 expression with methylation status of the gene

  • Investigate histone modifications at the SDHAF2 locus

• Controls and validation:

  • Include multiple family branches to account for genetic background effects

  • Utilize both SDHAF2 mutation analysis and linkage analysis for confirmation

This approach can help elucidate the molecular basis of the observed maternal imprinting phenomenon, where individuals inheriting SDHAF2 mutations maternally do not develop paragangliomas despite carrying the mutation .

How can researchers address weak or non-specific signals when using FITC-conjugated SDHAF2 antibodies?

To address weak or non-specific signals:

Additionally, comparison with non-FITC conjugated primary antibodies and appropriate secondary antibodies may help determine whether issues are related to the FITC conjugation or the antibody itself .

What are the key considerations for quantitative analysis of SDHAF2 immunofluorescence data?

For quantitative analysis of SDHAF2 immunofluorescence:

• Image acquisition parameters:

  • Maintain consistent exposure settings across all samples

  • Avoid pixel saturation

  • Collect Z-stacks for 3D analysis when appropriate

  • Include flat-field correction for uniform illumination

• Analysis approach:

  • Define clear regions of interest (ROIs)

  • Measure integrated intensity rather than mean intensity alone

  • Normalize to cell number or area

  • Compare signal to background ratio across samples

• Controls for quantification:

  • Include calibration standards for fluorescence intensity

  • Measure autofluorescence in unstained samples

  • Use internal reference markers

• Statistical considerations:

  • Analyze sufficient cell numbers (>100 per condition)

  • Apply appropriate statistical tests for data distribution

  • Consider biological vs. technical replicates

These guidelines ensure reliable quantification of SDHAF2 expression levels and subcellular distribution patterns in immunofluorescence experiments .

How should researchers interpret discrepancies between SDHAF2 protein detection and genetic analysis results?

When encountering discrepancies between SDHAF2 protein detection and genetic findings:

• Consider post-transcriptional regulation:

  • Assess mRNA levels via qRT-PCR or RNA-seq

  • Investigate microRNA regulation of SDHAF2

  • Examine protein stability and half-life

• Evaluate antibody specificity:

  • Test multiple antibodies targeting different epitopes

  • Perform peptide competition assays

  • Validate with recombinant SDHAF2 protein

• Assess mutation effects:

  • Certain mutations may affect epitope recognition without eliminating protein

  • Mutations might alter protein localization rather than expression

  • Some mutations could affect function without changing protein levels

• Technical considerations:

  • Different methodologies have varying sensitivity limits

  • Sample processing can affect protein preservation differentially

  • Consider mosaic expression patterns in tissues

• Biological implications:

  • Compensatory mechanisms may upregulate related proteins

  • Alternative splicing might produce variant proteins

  • Post-translational modifications could mask epitopes

This systematic approach helps reconcile apparently contradictory findings between molecular and immunological detection methods .

How can SDHAF2 antibodies contribute to understanding the relationship between SDH dysfunction and oncometabolite accumulation?

SDHAF2 antibodies can facilitate investigation of SDH dysfunction and oncometabolite relationships through:

• Correlation studies:

  • Quantify SDHAF2 expression using calibrated immunofluorescence

  • Measure succinate accumulation via metabolomic analysis

  • Establish direct relationships between SDHAF2 levels and metabolite profiles

• Functional studies:

  • Assess SDH activity in cells with varying SDHAF2 expression

  • Correlate enzyme activity with immunostaining intensity

  • Investigate SDHAF2-dependent changes in HIF-1α stabilization

• In situ approaches:

  • Perform co-staining of SDHAF2 with markers of hypoxic response

  • Analyze spatial relationships between SDHAF2 expression and pseudohypoxic signaling

  • Develop multiplexed assays for simultaneous detection of SDHAF2 and metabolic markers

• Intervention studies:

  • Monitor SDHAF2 levels during pharmacological interventions targeting oncometabolite effects

  • Assess therapeutic responses in relation to baseline SDHAF2 expression

These approaches can provide mechanistic insights into how SDHAF2 dysfunction contributes to the metabolic reprogramming observed in paragangliomas and other SDH-deficient tumors .

What methodological approaches can identify novel interaction partners of SDHAF2 using antibody-based techniques?

To identify novel SDHAF2 interaction partners:

• Proximity-dependent approaches:

  • BioID or TurboID fusion with SDHAF2 followed by streptavidin pull-down

  • APEX2-SDHAF2 fusion for proximity labeling of interaction partners

  • These methods identify proteins in proximity to SDHAF2 in living cells

• Co-immunoprecipitation strategies:

  • Stable isotope labeling (SILAC) combined with SDHAF2 immunoprecipitation

  • Chemical crosslinking prior to immunoprecipitation to capture transient interactions

  • Sequential immunoprecipitation (tandem IP) to isolate specific complexes

• Protein microarray screening:

  • Probe protein arrays with purified SDHAF2

  • Validate hits using reciprocal co-immunoprecipitation

• In situ approaches:

  • Proximity ligation assay (PLA) to screen candidate interactors

  • FRET-based screening with SDHAF2-fluorescent protein fusions

• Validation methods:

  • Recombinant protein binding assays

  • Mutational analysis of interaction domains

  • Functional assays to determine biological relevance

This comprehensive approach can reveal previously unknown SDHAF2 interactions beyond the established role in SDHA flavination .

How can researchers design experiments to study the temporal dynamics of SDHAF2 localization during mitochondrial biogenesis?

To study temporal dynamics of SDHAF2 localization during mitochondrial biogenesis:

• Inducible systems:

  • Create cell lines with fluorescently tagged SDHAF2 under inducible promoters

  • Develop FITC-conjugated antibodies compatible with live-cell imaging

  • Establish systems to trigger synchronized mitochondrial biogenesis

• Time-lapse imaging approaches:

  • Perform confocal time-lapse microscopy of tagged SDHAF2

  • Co-stain with mitochondrial markers during biogenesis

  • Use FRAP (Fluorescence Recovery After Photobleaching) to measure protein mobility

• Pulse-chase experimental design:

  • Perform temporal immunofluorescence studies at defined intervals

  • Combine with markers of different mitochondrial biogenesis stages

  • Correlate with markers of mitochondrial import machinery

• Quantitative analysis:

  • Measure colocalization coefficients over time

  • Track changes in subcellular distribution

  • Quantify protein accumulation rates in mitochondrial subcompartments

• Perturbation approaches:

  • Inhibit specific steps of mitochondrial biogenesis

  • Assess effects on SDHAF2 localization and dynamics

  • Compare wild-type and mutant SDHAF2 trafficking patterns

This methodological framework enables detailed characterization of SDHAF2's spatial and temporal dynamics during mitochondrial biogenesis and SDH complex assembly .

How do antibodies against different SDH complex components compare in their research applications?

Comparative analysis of antibodies against SDH components:

Combined use of antibodies against multiple complex components provides comprehensive insights into SDH complex assembly, stability, and function. Sequential or simultaneous immunolabeling approaches can reveal hierarchical assembly defects in patient samples or experimental models .

What considerations are important when designing multiplexed immunofluorescence experiments including SDHAF2?

For multiplexed immunofluorescence with SDHAF2:

• Antibody selection:

  • Choose primary antibodies from different host species

  • Consider directly conjugated antibodies with non-overlapping spectra

  • Validate each antibody individually before multiplexing

• Fluorophore selection:

  • For FITC-conjugated SDHAF2 (emission ~520nm), combine with:

    • Far-red fluorophores (>650nm) for maximum spectral separation

    • Orange/red fluorophores (580-620nm) for triple labeling

  • Account for spectral overlap and bleed-through

• Protocol optimization:

  • Test sequential vs. simultaneous antibody incubation

  • Optimize concentrations of each antibody separately

  • Consider tyramide signal amplification for low-abundance targets

• Controls for multiplexed imaging:

  • Single-color controls for spectral unmixing

  • Minus-one controls to assess bleed-through

  • Blocking controls between sequential applications

• Analysis considerations:

  • Apply appropriate spectral unmixing algorithms

  • Establish colocalization metrics and thresholds

  • Use appropriate statistical methods for correlation analysis

These guidelines ensure reliable detection of SDHAF2 alongside other proteins of interest in complex tissue or cellular samples .

How can SDHAF2 antibodies be utilized in high-content screening approaches?

For high-content screening with SDHAF2 antibodies:

• Assay development:

  • Optimize FITC-conjugated SDHAF2 antibody concentration for automated imaging

  • Establish cell models with varying SDHAF2 expression levels as controls

  • Develop automated image analysis pipelines for SDHAF2 quantification

• Screening applications:

  • Drug screens to identify compounds affecting SDHAF2 expression or localization

  • siRNA/CRISPR screens to identify regulators of SDHAF2

  • Chemical library screens to find modulators of SDH assembly

• Multiparametric analysis:

  • Combine SDHAF2 detection with:

    • Mitochondrial morphology measurements

    • Cell viability assessments

    • Metabolic activity indicators

    • Oxidative stress markers

• Technical considerations:

  • Implement automated liquid handling for consistent immunostaining

  • Develop robust cell segmentation algorithms

  • Establish quality control metrics for image and data quality

  • Apply machine learning approaches for multiparametric phenotype classification