C19orf12 Antibody

What is C19orf12 and why is it important in neurodegenerative research?

C19orf12 is a transmembrane protein associated with Neurodegeneration with Brain Iron Accumulation (NBIA), a clinical entity characterized by iron accumulation in the basal ganglia. The protein contains glycine zipper motifs forming helical regions that span the membrane. Understanding C19orf12 is critical for research into NBIA pathogenesis, particularly the variant known as MPAN (Mitochondrial membrane Protein-Associated Neurodegeneration) . The protein's importance lies in its dual localization in mitochondria and endoplasmic reticulum, suggesting multiple roles in cellular homeostasis.

What is the subcellular localization of wild-type C19orf12 protein?

Wild-type C19orf12 protein demonstrates multiple subcellular localizations. Western-blot analysis has confirmed that the protein is present in mitochondrial membranes, but also in the cellular lysate and cytosol. Further fractionation studies have revealed its presence in pure mitochondria, membrane-associated mitochondria (MAM), and endoplasmic reticulum (ER) . This diverse localization pattern suggests the protein may function at the interface between these organelles, particularly at the ER-mitochondria contact sites that are crucial for phospholipid transport and calcium signaling.

What applications are C19orf12 antibodies most commonly used for in research?

C19orf12 antibodies are predominantly used for:

• Western blot analysis to detect the protein in various subcellular fractions

• Immunodetection at 1:1000 dilution for specific C19orf12 protein visualization

• Subcellular localization studies to determine compartmentalization of wild-type and mutant proteins

• Comparative studies between patient-derived and control samples

• Monitoring protein response to oxidative stress conditions

The antibody has proven effective in distinguishing between normal localization patterns and aberrant ones caused by mutations, making it valuable for studying disease mechanisms.

What is the optimal protocol for using C19orf12 antibody in Western blot analysis?

For optimal Western blot analysis with C19orf12 antibody:

• Lyse cells using NP-40 cell lysis buffer (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na₃CO₄, and 1% NP40) supplemented with 1X protease inhibitor cocktail

• Centrifuge lysates at 18,000 g at 4°C for 30 minutes

• Separate proteins using 4-12% Bis-Tris Protein Gel

• Transfer to PVDF membrane

• Block membrane with appropriate blocking solution

• Incubate with C19orf12 primary antibody at 1:1000 dilution for 1-2 hours at room temperature

• Wash and apply appropriate secondary antibody

• Visualize using standard chemiluminescence methods

This protocol has been validated for detecting both wild-type and mutant forms of C19orf12 protein in various cellular compartments.

How can researchers effectively isolate subcellular fractions to study C19orf12 localization?

To effectively isolate subcellular fractions for C19orf12 localization studies:

• Begin with HEK293 or similar cells expressing C19orf12 (endogenous or tagged)

• Prepare separate fractions containing:

  • Crude mitochondria

  • Pure mitochondria (through gradient purification)

  • Membrane-associated mitochondria (MAM)

  • Endoplasmic reticulum

• Validate fraction purity using established markers:

  • Inositol 3 Phosphate receptor 3 (IP3R3) for ER

  • Tubulin for cytoplasm

  • VDAC for mitochondria

• Perform Western blot with C19orf12 antibody (1:1000 dilution)

• Compare distributions between wild-type and mutant proteins

This fractionation approach revealed the novel finding that C19orf12 localizes to multiple cellular compartments, which was previously unknown.

What controls should be included when using C19orf12 antibody for immunodetection?

When using C19orf12 antibody for immunodetection, the following controls are essential:

• Positive controls:

  • Overexpressed wild-type C19orf12 (tagged or untagged)

  • Known positive cell lines (such as HeLa or HEK293 with confirmed expression)

• Negative controls:

  • C19orf12 knockdown or knockout samples

  • Secondary antibody-only controls

• Loading and fractionation controls:

  • NDUFA9 antibody (0.5 μg/ml) for mitochondrial proteins

  • β-TUBULIN antibody (1 μg/ml) for cytosolic proteins

  • IP3R3 antibody (1:300) for ER proteins

  • VDAC antibody (1:3000) for mitochondrial membrane proteins

• Expression level controls:

  • Real-time PCR to quantify overexpression levels relative to endogenous C19orf12

How do mutations in C19orf12 affect antibody detection and protein localization?

Mutations in C19orf12 significantly alter protein localization, which affects antibody detection patterns in different subcellular fractions:

• Wild-type C19orf12: Primarily detected in mitochondrial membranes, ER, and MAM fractions with distinctive network-like intracellular staining pattern

• G58S mutation: This mutation affects the glycine zipper transmembrane domain, causing the protein to mislocalize partially to the mitochondrial matrix and cytosol. Western blot analysis shows diffuse cytosolic distribution rather than membrane association. Confocal microscopy reveals asymmetric colocalization patterns with organelle markers.

• Q96P mutation: While this mutation preserves mitochondrial membrane localization similar to wild-type, it creates an abnormal vesicular pattern rather than the normal network distribution. Colocalization analysis shows reduced Pearson's coefficients with both mitochondrial and ER markers .

These altered localization patterns provide insights into structure-function relationships of C19orf12 and can be leveraged for studying pathogenic mechanisms.

What approaches can be used to study the dynamic relocalization of C19orf12 under oxidative stress?

To study C19orf12 dynamic relocalization under oxidative stress:

• Live cell imaging approach:

  • Transfect cells with fluorescently tagged C19orf12 constructs (GFP or mKate2)

  • Establish baseline localization with organelle markers (mitotracker, ER markers)

  • Apply oxidative stress (e.g., H₂O₂ treatment)

  • Perform time-lapse imaging to capture protein movement

  • Quantify redistribution using colocalization statistics (Pearson's and Mander's coefficients)

• Biochemical fractionation approach:

  • Treat cells with oxidative stressors at varying timepoints

  • Perform subcellular fractionation

  • Quantify C19orf12 levels in each fraction by Western blot

  • Compare wild-type versus mutant behavior

• Functional correlation:

  • Monitor autophagy markers (LC3, p62) simultaneously

  • Track mitochondrial calcium levels

  • Assess apoptotic markers to correlate relocalization with functional outcomes

Research has shown that wild-type C19orf12 responds to oxidative stress by relocating to the cytosol and forming aggregates, while mutant proteins fail to respond, suggesting impaired stress response mechanisms in disease states.

How can C19orf12 antibodies be used to investigate the protein's role in autophagy regulation?

C19orf12 antibodies can be strategically employed to investigate the protein's role in autophagy regulation through several approaches:

• Colocalization studies:

  • Perform double immunostaining with C19orf12 antibody and autophagy markers (LC3, p62)

  • Analyze spatial relationships during basal conditions and after autophagy induction

  • Quantify changes in colocalization patterns

• Autophagy flux analysis:

  • Compare LC3-I to LC3-II conversion in cells with different C19orf12 status (wild-type, overexpressed, mutant)

  • Monitor p62 degradation rates in correlation with C19orf12 levels

  • Use C19orf12 antibody to confirm expression levels in parallel

• Structure-function relationships:

  • Generate domain-specific C19orf12 constructs

  • Use antibodies to verify expression and localization of truncated proteins

  • Correlate structural features with autophagy modulation capacity

• Temporal dynamics:

  • Track C19orf12 relocalization during different stages of autophagy

  • Correlate with the formation and dissolution of LC3-positive vesicles

Research has shown that wild-type C19orf12 overexpression promotes LC3 conversion and reduces p62 levels, suggesting autophagy activation, while mutant proteins fail to induce these changes.

What are the key considerations for distinguishing between wild-type and mutant C19orf12 proteins using antibodies?

Distinguishing between wild-type and mutant C19orf12 proteins requires careful experimental design:

• Antibody selection:

  • Standard C19orf12 antibodies typically recognize both wild-type and mutant forms

  • When possible, use mutation-specific antibodies for direct detection of variants

• Fractionation approach:

  • G58S mutation causes redistribution to mitochondrial matrix and cytosol

  • Q96P mutation maintains membrane localization but with abnormal vesicular patterns

  • Careful subcellular fractionation can help distinguish localization differences

• Molecular weight considerations:

  • Wild-type C19orf12 is a 17 kDa protein

  • Some mutations may cause subtle mobility shifts

  • Use high-resolution gels (15-20%) for optimal separation

• Live imaging complementation:

  • Fluorescently tagged constructs can help visualize distribution patterns

  • Wild-type shows network-like patterns

  • G58S shows cytosolic distribution

  • Q96P displays vesicular patterns

Combining biochemical fractionation with imaging approaches provides the most comprehensive discrimination between wild-type and mutant forms.

How can researchers optimize immunodetection of low-abundance C19orf12 in patient-derived samples?

For optimizing detection of low-abundance C19orf12 in patient samples:

• Sample preparation optimization:

  • Use specialized lysis buffers with multiple protease inhibitors

  • Perform protein concentration step if necessary

  • Consider subcellular fractionation to enrich for C19orf12-containing compartments

• Signal amplification strategies:

  • Employ high-sensitivity chemiluminescent substrates

  • Consider using HRP-conjugated polymers instead of standard secondary antibodies

  • Optimize antibody concentration through titration experiments

• Loading considerations:

  • Maximize protein loading (50-100 μg per lane)

  • Use larger wells for better resolution

  • Consider concentration of samples through immunoprecipitation prior to Western blot

• Extended exposure optimization:

  • Use incremental exposure times

  • Employ cooled CCD camera systems for better signal-to-noise ratio

  • Consider computational enhancement of weak signals

• Technical alternatives:

  • For extremely low abundance, consider mass spectrometry-based approaches

  • Use proximity ligation assay for improved sensitivity in tissue sections

These approaches can help detect physiological levels of C19orf12 in patient fibroblasts or other clinical samples where expression may be limited.

What experimental approaches can validate C19orf12 antibody specificity in various applications?

To validate C19orf12 antibody specificity across applications:

• Genetic validation:

  • Compare detection in wild-type versus C19orf12 knockout/knockdown models

  • Overexpression systems with tagged variants for antibody validation

  • Real-time PCR correlation with protein levels detected by antibody

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Compare signal between blocked and unblocked antibody

  • Gradual reduction in signal with increasing peptide concentration confirms specificity

• Multiple antibody concordance:

  • Compare detection patterns using antibodies raised against different epitopes

  • Consistent patterns across antibodies support specificity

  • Discordant results warrant further investigation

• Cross-species reactivity assessment:

  • Test antibody against C19orf12 from different species

  • Expected conservation patterns should match detection patterns

  • Helps establish evolutionary conservation of epitopes

• Signal validation in diverse applications:

  • Compare patterns in Western blot, immunocytochemistry, and immunoprecipitation

  • Consistent findings across methods support specificity

Thorough validation is essential when studying proteins like C19orf12 where disease-causing mutations may affect antibody binding properties.

How can C19orf12 antibodies be used to investigate the protein's potential role in magnesium transport regulation?

Given the structural homology between C19orf12 and bacterial MgtE transporters, researchers can use C19orf12 antibodies to investigate magnesium transport regulation:

• Co-immunoprecipitation studies:

  • Use C19orf12 antibodies to pull down protein complexes

  • Analyze for presence of human MgtE-like transporters

  • Identify interaction partners involved in magnesium homeostasis

• Functional assays:

  • Measure intracellular magnesium levels in cells with varying C19orf12 status

  • Correlate C19orf12 expression (verified by antibody) with magnesium transport capacity

  • Assess impact of C19orf12 mutations on magnesium handling

• Structure-function correlation:

  • Generate domain-specific deletions in the regulatory domain of C19orf12

  • Use antibodies to confirm expression levels

  • Correlate structural integrity with magnesium transport regulation

• Disease-relevant models:

  • Analyze magnesium levels in MPAN patient-derived cells

  • Correlate with C19orf12 localization and expression

  • Investigate potential therapeutic approaches targeting magnesium homeostasis

This research direction is particularly relevant since magnesium deficiency has been implicated in Parkinson's and other neurodegenerative diseases, potentially connecting C19orf12 dysfunction to broader neurodegeneration mechanisms.

What approaches can be used to study the relationship between C19orf12 and ferroptosis using specific antibodies?

To investigate C19orf12's role in ferroptosis using antibodies:

• Expression correlation studies:

  • Use C19orf12 antibodies to quantify protein levels during ferroptosis induction

  • Compare wild-type versus C19orf12-deficient cells for ferroptosis markers

  • Monitor temporal dynamics of expression changes

• Oxidative stress markers co-detection:

  • Perform dual immunodetection of C19orf12 and oxidative stress markers (8OHG, HNE)

  • Compare wild-type and mutant C19orf12 effects on oxidative stress marker levels

  • Correlate with ferroptosis progression

• Pathway component analysis:

  • Monitor key ferroptosis proteins (HMOX1, GPX4) in relation to C19orf12 status

  • Use C19orf12 antibodies to confirm knockdown/overexpression efficiency

  • Assess whether C19orf12 acts upstream or downstream of established ferroptosis regulators

• Lipidomic correlation:

  • Quantify lipid peroxidation products in cells with different C19orf12 expression

  • Correlate C19orf12 levels (detected by antibody) with specific lipid species altered in ferroptosis

  • Determine if C19orf12 directly impacts lipid metabolism pathways involved in ferroptosis

These approaches leverage C19orf12 antibodies to establish the mechanistic connection between this protein and iron-dependent regulated cell death, which appears particularly relevant to its role in neurodegeneration with brain iron accumulation.

How can researchers best combine C19orf12 antibodies with calcium imaging to study mitochondrial calcium dynamics?

To effectively combine C19orf12 antibody studies with calcium imaging for investigating mitochondrial calcium dynamics:

• Sequential experimental design:

  • Perform live calcium imaging using fluorescent indicators (Rhod-2, Fluo-4)

  • Fix the same cells and immunostain for C19orf12

  • Correlate calcium handling with C19orf12 distribution patterns

• Genetic modification approach:

  • Create stable cell lines with varying C19orf12 status (overexpression, knockdown, mutations)

  • Confirm protein levels/localization using antibodies

  • Perform calcium imaging experiments under various conditions

  • Compare responses between genetically defined populations

• Combined microscopy techniques:

  • Use split-channel imaging to simultaneously monitor calcium indicators and fluorescently tagged C19orf12

  • Validate findings with fixed-cell immunocytochemistry using C19orf12 antibodies

  • Analyze correlation between C19orf12 localization and calcium microdomains

• ER-mitochondria contact site focus:

  • Use C19orf12 antibodies to quantify protein enrichment at MAM regions

  • Correlate with calcium transfer efficiency between ER and mitochondria

  • Investigate impact of C19orf12 mutations on contact site integrity and function

This combined approach is particularly valuable since patient fibroblasts with C19orf12 mutations show elevated mitochondrial calcium levels and increased sensitivity to calcium-dependent apoptotic stimuli like H₂O₂.

How do C19orf12 antibody detection patterns differ between various neurodegeneration models?

C19orf12 antibody detection patterns show distinctive characteristics across neurodegeneration models:

Understanding these differential patterns helps researchers select appropriate detection methods and experimental designs when using C19orf12 antibodies in disease models .

What is the relationship between C19orf12 mutations, protein mislocalization, and mitochondrial dysfunction?

The relationship between C19orf12 mutations, protein mislocalization, and mitochondrial dysfunction follows a complex pattern:

• Structure-localization correlation:

  • G58S mutation in the glycine zipper transmembrane domain causes mislocalization to the mitochondrial matrix and cytosol

  • Q96P mutation in the regulatory domain creates abnormal vesicular patterns

  • Both mutations impair the protein's normal distribution at ER-mitochondria contact sites

• Functional consequences:

  • Patient fibroblasts with C19orf12 mutations show:

    • Elevated mitochondrial calcium levels

    • Increased sensitivity to H₂O₂-induced apoptosis

    • Impaired autophagy induction

    • Potential accumulation of dysfunctional mitochondria

• Stress response defects:

  • Wild-type C19orf12 relocates to cytosol under oxidative stress

  • Mutant proteins fail to respond to stress conditions

  • This suggests an impaired mitochondrial quality control mechanism

• Potential mechanisms:

  • Disruption of ER-mitochondria communication

  • Impaired calcium homeostasis

  • Defective mitophagy

  • Possible interference with magnesium transport regulation

These relationships can be detected and characterized using C19orf12 antibodies in fractionation studies, providing insight into disease pathogenesis.

How can researchers reconcile conflicting data about C19orf12 function and localization from different studies?

To reconcile conflicting data about C19orf12 function and localization:

• Methodological differences assessment:

  • Compare antibody sources, clones, and epitopes across studies

  • Evaluate cell types used (patient-derived vs. models)

  • Assess fractionation protocols (crude vs. pure preparations)

  • Consider detection methods (Western blot vs. microscopy)

• Experimental condition reconciliation:

  • Analyze baseline vs. stress conditions

  • Consider timing of observations (acute vs. chronic)

  • Evaluate expression level differences (endogenous vs. overexpressed)

  • Assess the impact of tags (size, position) on protein behavior

• Unified model development:

  • C19orf12 shows dual localization (mitochondria and ER)

  • Dynamic redistribution occurs under stress

  • Different mutations affect distinct protein domains and functions

  • Protein likely has multiple roles depending on cellular context

• Technical validation approaches:

  • Cross-validate using multiple antibodies

  • Employ complementary techniques (biochemical + imaging)

  • Use genetic models with defined C19orf12 status

  • Consider post-translational modifications that might affect detection

The apparent contradictions may reflect the dynamic nature of C19orf12 biology, with different studies capturing distinct states of this multifunctional protein.