C19orf12 Antibody, HRP conjugated

What is C19orf12 and why is it significant in neurodegeneration research?

C19orf12 is a 17 kDa transmembrane protein associated with Mitochondrial membrane protein-associated neurodegeneration (MPAN), a rare form of Neurodegeneration with Brain Iron Accumulation (NBIA). This protein is found in mitochondria, Endoplasmic Reticulum (ER), and Mitochondria Associated Membrane (MAM). Mutations in the C19orf12 gene have been identified in patients with NBIA, making it a crucial target for understanding the pathophysiology of iron accumulation disorders .

Methodological approach: When investigating C19orf12's role in neurodegeneration, researchers should combine genetic analysis (to identify mutations), protein localization studies using specific antibodies, and functional assays to assess mitochondrial function and oxidative stress responses in patient-derived cells or model systems. Subcellular fractionation followed by western blot analysis with appropriate markers (IP3R3 for ER, VDAC for mitochondria) provides valuable insights into protein distribution .

What are the known structural features of C19orf12 and how do they relate to antibody selection?

C19orf12 contains glycine zipper motifs that form helical regions spanning the membrane. The N- and C-terminal regions form a structural domain homologous to the N-terminal regulatory domain of magnesium transporter MgtE, suggesting C19orf12 may function as a regulatory protein for human MgtE transporters .

For antibody selection, researchers should consider:

• Epitope location: The HRP-conjugated C19orf12 antibody (ABIN7165169) targets amino acids 65-104

• Transmembrane regions: Antibodies targeting extramembrane domains typically perform better in applications like immunofluorescence

• Mutation locations: The G58S mutation affects a glycine residue in the transmembrane zipper motifs, while Q96P affects the regulatory domain

How does the subcellular localization of wild-type C19orf12 differ from mutant variants?

Wild-type C19orf12 exhibits a complex distribution pattern that changes under specific conditions:

This differential localization can be quantified using Pearson's and Mander's coefficients to measure correlation between C19orf12 signal and organelle markers .

What are the optimal applications for HRP-conjugated C19orf12 antibody?

The HRP-conjugated C19orf12 antibody is particularly well-suited for:

• ELISA: Primary application as indicated by manufacturer

• Western blot analysis: For detecting C19orf12 in subcellular fractions (typically using 30μg protein per lane)

• Immunohistochemistry: HRP conjugation eliminates need for secondary antibody incubation

Methodological considerations:

• Working dilution should be empirically determined for each application

• Storage at -20°C or -80°C, avoiding repeated freeze-thaw cycles

• Contains ProClin preservative which requires careful handling as a hazardous substance

How should researchers prepare samples for C19orf12 detection in different subcellular fractions?

Optimized sample preparation protocol:

• Subcellular fractionation:

  • Use differential centrifugation to isolate mitochondria, ER, MAM, and cytosolic fractions

  • Verify fraction purity using established markers: IP3R3 for ER, VDAC for mitochondria, tubulin for cytoplasm

• Sample processing:

  • Load 30μg of protein per lane for western blot analysis

  • Use denaturing SDS-PAGE for separation

  • Transfer to appropriate membrane for western blot detection

• Antibody application:

  • Use anti-C19orf12 antibody at 1:1000 dilution

  • For HRP-conjugated antibody, proceed directly to detection

  • For unconjugated antibody, use secondary anti-rabbit antibody at 1:7000 dilution

How can C19orf12 antibodies be used to investigate oxidative stress responses in NBIA models?

Advanced experimental approach:

• Baseline characterization:

  • Establish normal C19orf12 localization patterns in control and patient-derived cells

  • Validate antibody specificity with appropriate controls

• Oxidative stress protocol:

  • Treat cells with H₂O₂ (500μM) for temporal analysis

  • Monitor protein redistribution using live cell imaging (if using fluorescently-tagged constructs) or fixed timepoint analysis with C19orf12 antibody

• Quantitative analysis strategy:

  • Track C19orf12 redistribution from reticular pattern to cytosolic localization (occurs after ~30 minutes of H₂O₂ exposure)

  • Measure formation of bright aggregates partially colocalizing with mitochondria during persistent stress

  • Compare responses between wild-type and mutant C19orf12 variants

Research findings indicate wild-type C19orf12 responds to oxidative stress by relocating to the cytosol and forming aggregates, while mutant variants (G58S and Q96P) fail to undergo this relocalization, suggesting impaired stress response mechanisms .

What methodological approaches can be used to study the potential role of C19orf12 as a regulatory protein for MgtE transporters?

Comprehensive research strategy:

• Structural analysis:

  • Utilize in silico modeling of C19orf12 based on homology to N-terminal regulatory domain of bacterial MgtE transporters

  • Identify conserved functional residues through sequence alignment

  • Predict structural impact of disease-causing mutations:

    • G58S affects glycine zipper motifs involved in transmembrane helix dimerization

    • Q96P may disrupt helical structure in the regulatory domain important for protein-protein interactions

• Protein-protein interaction studies:

  • Co-immunoprecipitation using C19orf12 antibody to pull down potential MgtE transporter interactions

  • Proximity ligation assays for in situ detection of interactions

  • Functional assays measuring magnesium transport in the presence of wild-type vs. mutant C19orf12

How can researchers optimize co-localization studies to investigate C19orf12's role in mitochondria-ER communication?

Advanced co-localization methodology:

• Multi-channel confocal microscopy setup:

  • Channel 1: C19orf12 antibody or fluorescently-tagged construct

  • Channel 2: Mitochondrial marker (e.g., mitotracker Deep Red)

  • Channel 3: ER marker (e.g., GFP–Sec61-β)

  • Channel 4: Additional markers for MAM or other relevant structures

• Quantitative analysis framework:

  • Global colocalization coefficients:

    • Pearson's correlation coefficient: Measures correlation between signals

    • Mander's overlap coefficient: Proportion of C19orf12 signal overlapping with organelle markers

• Functional readouts:

  • Mitochondrial calcium measurements (higher in patient fibroblasts compared to controls)

  • H₂O₂-induced apoptosis (increased in patient cells)

  • Changes in mitochondria-ER contact sites under different conditions

Findings from published research demonstrate that wild-type C19orf12 shows significant colocalization with both ER and mitochondrial markers, while mutations alter this pattern, potentially disrupting inter-organelle communication .

What considerations should be made when designing experiments to compare wild-type and mutant C19orf12 function?

Experimental design considerations:

• Expression system options:

  • Endogenous detection: Direct use of C19orf12 antibody on patient-derived cells

  • Tagged systems: Myc-tagged or fluorescently-tagged (GFP, mKate2) constructs for live imaging

  • Control for expression levels: Use Real-time PCR to quantify expression of transfected constructs

• Mutation analysis strategy:

  • G58S: Located in predicted transmembrane domain, causes mislocalization to mitochondrial matrix

  • Q96P: Located in regulatory domain, alters distribution to vesicular pattern

  • Both mutations impair response to oxidative stress

• Critical controls:

  • Include wild-type construct in parallel experiments

  • Validate subcellular fractionation with appropriate markers

  • Perform colocalization quantification using standardized methods

How can researchers investigate the relationship between C19orf12 mutations and altered calcium homeostasis in NBIA?

Methodological approach:

• Calcium measurement techniques:

  • Use calcium-sensitive fluorescent indicators to monitor subcellular calcium concentrations

  • Compare baseline and stress-induced calcium levels between control and patient-derived cells

• Mitochondrial function assessment:

  • Measure mitochondrial membrane potential

  • Assess respiratory chain function

  • Quantify H₂O₂-induced apoptosis rates

• MAM integrity analysis:

  • Evaluate physical connections between mitochondria and ER

  • Measure functional readouts of MAM activity (lipid transfer, calcium signaling)

  • Assess impact of C19orf12 mutations on MAM structure and function

Research findings indicate that patient fibroblasts with C19orf12 mutations show elevated mitochondrial calcium concentrations and increased susceptibility to H₂O₂-induced apoptosis, suggesting disruption of calcium homeostasis as a potential pathogenic mechanism in MPAN .