C12orf65 Antibody

What is C12orf65 and what cellular functions does it serve?

C12orf65 (Chromosome 12 Open Reading Frame 65) encodes a mitochondrial matrix protein that plays a crucial role in releasing peptides from mitochondrial ribosomes during translation. It participates extensively in mitochondrial protein synthesis and is essential for proper mitochondrial function. The protein contains 166 amino acids with a calculated molecular weight of approximately 19 kDa. Functionally, C12orf65 ensures proper termination of mitochondrial translation, and its dysfunction leads to global decrease in mitochondrial protein synthesis without affecting mitochondrial RNA levels.

What disease associations have been identified with C12orf65 mutations?

Mutations in C12orf65 have been associated with several neurological disorders that form a clinical spectrum. The key phenotypes include:

Additionally, C12orf65 mutations can cause combined oxidative phosphorylation deficiency type 7 (COXPD7) and autosomal recessive spastic paraplegia 55 (SPG55). There is an emerging genotype-phenotype correlation where mutations disrupting the GGQ-containing domain in the first coding exon typically result in more severe phenotypes, while downstream C-terminal mutations may result in milder phenotypes without cognitive impairment.

What are the basic specifications of commercially available C12orf65 antibodies?

The C12orf65 antibody (such as the 24646-1-AP variant) is typically a rabbit polyclonal IgG antibody that targets human C12orf65 protein. The antibody is generated using C12orf65 fusion protein as the immunogen. It is supplied in liquid form, purified through antigen affinity purification, and stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. The antibody has been validated for immunohistochemistry (IHC) and ELISA applications, with demonstrated reactivity against human samples. For IHC applications, the recommended dilution range is 1:50-1:500.

How does C12orf65 deficiency affect mitochondrial translation at the molecular level?

C12orf65 deficiency leads to a global decrease in mitochondrial protein synthesis as demonstrated by metabolic labeling studies with [35S]-methionine/cysteine. Interestingly, this reduction in protein synthesis is not due to decreased mitochondrial RNA levels, which are actually modestly elevated in patient cells with C12orf65 mutations. When C12orf65 is artificially depleted using siRNA, the elevation in mitochondrial RNA levels becomes even more pronounced, suggesting a compensatory mechanism.

What are the critical considerations when designing experiments to study the effects of C12orf65 knockdown or knockout?

When designing experiments to study C12orf65 function through knockdown or knockout approaches, researchers should consider:

• Cell type selection: Different cell types exhibit varying dependence on mitochondrial function. Neurons, muscle cells, and retinal cells are particularly sensitive to mitochondrial dysfunction, making them suitable models for studying C12orf65-related disorders.

• Knockdown approach: siRNA targeting C12orf65 has been successfully used (e.g., sense 5′-GGG AGA AGC UGA CGU UGU U dTdT) at 30nM final concentration. Complete knockout may be lethal, so inducible systems might be preferable for long-term studies.

• Phenotypic readouts: Multiple assays should be employed to capture the range of effects:

  • Metabolic labeling of mitochondrially encoded proteins with [35S]-methionine/cysteine

  • Northern blot analysis of mitochondrial RNA levels

  • Blue Native PAGE for respiratory complex assembly

  • Mitochondrial respiration measurements

  • Analysis of mitoribosomal protein levels by western blotting

• Controls: Include both non-targeting siRNA controls and rescue experiments with wild-type C12orf65 to confirm specificity.

How do different mutations in C12orf65 correlate with phenotypic severity and biochemical defects?

Research has established an emerging genotype-phenotype correlation for C12orf65 mutations:

The frameshift mutation c.171_172delGA (p.N58fs) has been studied in detail, including autopsy findings revealing symmetrical cyst formation with brownish lesions in the upper spinal cord, ventral medulla, pons, dorsal midbrain, and medial hypothalamus. Microscopic analysis of these areas demonstrated mild gliosis with rarefaction, while electron microscopy revealed numerous abnormal mitochondria in the choroid plexus cells.

What are the optimal conditions for using C12orf65 antibodies in immunohistochemistry?

For optimal immunohistochemistry (IHC) results with C12orf65 antibodies:

• Dilution range: Use the antibody at 1:50-1:500 dilution, with the optimal concentration determined through titration for each specific testing system and sample type.

• Antigen retrieval: For human kidney tissue and other samples, two methods are recommended:

  • Primary method: Tris-EDTA (TE) buffer at pH 9.0

  • Alternative method: Citrate buffer at pH 6.0

• Detection system: Standard secondary antibody detection systems compatible with rabbit IgG are suitable.

• Positive control: Human kidney tissue has been validated as a positive control for C12orf65 antibody staining.

• Storage and handling: Store the antibody at -20°C where it remains stable for one year after shipment. Aliquoting is not necessary for -20°C storage. Formulations containing 0.1% BSA (in 20μl sizes) provide additional stability.

How can researchers effectively validate the specificity of C12orf65 antibodies in their experimental systems?

To validate C12orf65 antibody specificity:

• Positive and negative tissue controls: Use tissues with known C12orf65 expression patterns (e.g., human kidney as positive control) and tissues from species not cross-reactive with the antibody as negative controls.

• siRNA knockdown validation: Perform siRNA-mediated knockdown of C12orf65 in cell culture and confirm reduced antibody signal by western blot or immunostaining. The following siRNA sequence has been effectively used: sense 5′-GGG AGA AGC UGA CGU UGU U dTdT at 30nM concentration.

• Overexpression studies: Express tagged versions of C12orf65 and confirm co-localization with the antibody signal.

• Western blot analysis: Confirm detection of a protein band at the expected molecular weight (19 kDa), with reduced intensity in knockdown samples.

• Mitochondrial fractionation: Since C12orf65 is a mitochondrial protein, confirm enrichment of the signal in mitochondrial fractions compared to cytosolic fractions.

What techniques can be used to assess the functional impacts of C12orf65 mutations?

Multiple complementary techniques can assess the functional impact of C12orf65 mutations:

What are common challenges when using C12orf65 antibodies for IHC, and how can they be addressed?

Common challenges and solutions for C12orf65 antibody use in IHC include:

Always titrate the antibody for each specific testing system to determine optimal conditions, as suggested in the product information.

How can researchers distinguish between primary C12orf65 dysfunction and secondary mitochondrial defects?

Distinguishing primary C12orf65 dysfunction from secondary mitochondrial defects requires a multi-faceted approach:

• Genetic analysis: Confirm pathogenic variants in C12orf65 through sequencing. Reported mutations include frameshift mutations (c.413_417 delAACAA, c.171_172delGA) and splice site mutations (g.21043 T>A, c.282+2 T>A) that lead to protein truncation or exon skipping.

• Molecular characterization:

  • C12orf65 deficiency shows a specific pattern of global decrease in mitochondrial protein synthesis with elevated mitochondrial RNA levels

  • No change in steady-state levels of mitoribosomal proteins

  • More severe effects on Complex IV activity compared to other complexes

• Rescue experiments:

  • Transfect cells with wild-type C12orf65 and assess restoration of mitochondrial translation

  • A primary C12orf65 defect will show normalization of translation patterns

• Clinical correlation:

  • Primary C12orf65 dysfunction typically presents with a triad of optic atrophy, axonal neuropathy, and spastic paraparesis

  • Other mitochondrial disorders may have overlapping but distinct clinical features

• Tissue specificity analysis:

  • C12orf65 mutations show characteristic patterns of central nervous system involvement, including symmetrical cyst formation with brownish lesions in specific brain regions as revealed in autopsy studies

What controls should be included when analyzing C12orf65 expression or function in experimental studies?

To ensure robust and interpretable results when studying C12orf65:

• Positive tissue controls:

  • Human kidney tissue has been validated for C12orf65 antibody staining

  • Include tissues with known high mitochondrial content (e.g., heart, skeletal muscle)

• Negative controls:

  • For antibody studies: secondary antibody-only controls, isotype controls, and tissues from non-cross-reactive species

  • For functional studies: non-targeting siRNA controls

• Knockdown/Knockout verification:

  • Confirm C12orf65 reduction at both mRNA and protein levels

  • Custom synthesized siRNA targeting C12orf65 (sense 5′-GGG AGA AGC UGA CGU UGU U dTdT) at 30nM has been validated

• Functional controls:

  • Include known mitochondrial translation inhibitors (e.g., chloramphenicol) as positive controls

  • For respiratory chain analysis, include specific inhibitors for each complex

• Restoration controls:

  • Complementation with wild-type C12orf65 to verify phenotype rescue

  • Complementation with mutant variants to confirm pathogenicity of specific mutations

• Mitochondrial markers:

  • Include analysis of mitochondrial mass markers (e.g., HSP60) to normalize for mitochondrial content

  • Monitor other mitochondrial processes (e.g., fusion/fission) that might secondarily affect translation

How can C12orf65 antibodies be used to investigate its role in different tissue types and disease states?

C12orf65 antibodies can be employed across multiple research applications to investigate tissue-specific roles and disease associations:

• Comparative tissue analysis:

  • Perform IHC across multiple tissue types to establish differential expression patterns

  • Pay special attention to tissues affected in C12orf65-related disorders (retina, brain, peripheral nerves, muscle)

  • Recommended dilution of 1:50-1:500 for IHC applications, optimized per tissue type

• Disease model characterization:

  • Compare C12orf65 expression and localization in normal versus disease tissues

  • Investigate tissues from patients with mitochondrial disorders of unknown etiology

  • Examine autopsy samples for pathological changes similar to those observed in confirmed cases (symmetrical cyst formation in brainstem regions)

• Co-localization studies:

  • Combine C12orf65 antibodies with markers for mitochondrial subcompartments to determine precise localization

  • Co-stain with mitoribosomal proteins to investigate functional interactions

• Developmental expression patterns:

  • Track C12orf65 expression during development to correlate with onset of disease symptoms

  • Particularly relevant for understanding early-onset optic atrophy and developmental delay

• Mutation impact analysis:

  • Express tagged mutant variants and examine cellular localization and stability

  • Compare experimental findings with clinical severity to validate genotype-phenotype correlations

What recent advances have been made in understanding the structure-function relationship of C12orf65?

Recent structural and functional studies of C12orf65 have revealed:

• Functional domains:

  • The GGQ-containing domain in the first coding exon is critical for peptide release activity during mitochondrial translation

  • C-terminal regions appear less critical for basic function but may mediate protein interactions or stability

  • Mutations disrupting the GGQ domain result in more severe clinical phenotypes than C-terminal mutations

• Evolutionary conservation:

  • C12orf65 is evolutionarily related to class I peptide release factors

  • The protein shares functional similarities with ICT1, another mitochondrial translation release factor

• Mutation consequences:

  • Frameshift mutations (e.g., c.413_417 delAACAA) result in truncated proteins lacking the C-terminal portion

  • Splice mutations (e.g., g.21043 T>A, c.282+2 T>A) lead to exon skipping

  • Truncated C12orf65 proteins may escape nonsense-mediated decay, suggesting they retain some function or acquire dominant-negative properties

• Tissue specificity:

  • Despite ubiquitous expression, mutations affect specific tissues (brain, retina, peripheral nerves)

  • This suggests tissue-specific roles or variable dependence on C12orf65 function

  • Autopsy findings have revealed characteristic lesions in specific brain regions

• Therapeutic implications:

  • Understanding domain-specific functions may enable targeted therapies

  • Identification of bypass mechanisms for defective mitochondrial peptide release could provide therapeutic strategies

How does C12orf65 dysfunction contribute to the pathophysiology of associated neurological disorders?

The pathophysiological mechanisms linking C12orf65 dysfunction to neurological disorders include:

• Mitochondrial translation defects:

  • Global decrease in mitochondrial protein synthesis

  • More severe impact on specific respiratory chain complexes (particularly Complex IV)

  • These defects lead to energy production deficits critical in high-energy demanding tissues

• Tissue-specific vulnerability:

  • Neurons, especially those with long axons (optic nerve, corticospinal tracts, peripheral nerves), have high energy demands

  • These tissues show preferential involvement despite the ubiquitous expression of C12orf65

  • Autopsy findings demonstrate specific vulnerability patterns with symmetrical cyst formation in the upper spinal cord, ventral medulla, pons, dorsal midbrain, and medial hypothalamus

• Biochemical abnormalities:

  • Elevated lactate levels in serum and CSF indicate impaired oxidative phosphorylation

  • Decreased activities of respiratory chain complexes I and IV in muscle biopsies

  • These changes reflect compromised energy metabolism

• Cellular pathology:

  • Electron microscopy reveals abnormal mitochondria, particularly in choroid plexus cells

  • Microscopic analysis of affected brain regions shows mild gliosis with rarefaction

  • These changes suggest chronic energy deficiency and cellular degeneration

• Clinical progression:

  • The slowly progressive nature of symptoms (especially peripheral neuropathy and spastic paraparesis) suggests cumulative damage

  • Early onset optic atrophy indicates particular vulnerability of retinal ganglion cells

  • The constellation of symptoms mirrors other mitochondrial translation disorders, supporting a shared pathophysiological mechanism