COX7C Antibody

What is COX7C and why is it important in cellular research?

COX7C is a nuclear-encoded subunit of cytochrome c oxidase (COX), the terminal component of the mitochondrial respiratory chain. This enzyme catalyzes the electron transfer from reduced cytochrome c to oxygen, creating an electrochemical gradient essential for ATP synthesis. COX7C is significant in research as it functions in the regulation and assembly of the complex, with the mitochondrially-encoded subunits primarily functioning in electron transfer . The protein is found in all tissues and shares high sequence identity (87% and 85%) with mouse and bovine COX VIIc, respectively .

What applications are COX7C antibodies most commonly used for?

COX7C antibodies are primarily employed in Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF)/Immunocytochemistry (ICC) applications. Based on validation data, these antibodies show reactivity with human, mouse, and rat samples . The recommended dilutions vary by application:

It's important to note that optimal dilutions should be determined for each specific experimental system .

What is the expected molecular weight for COX7C detection in Western blots?

While the calculated molecular weight of COX7C is approximately 7 kDa (63 amino acids), the observed molecular weight in Western blot experiments typically ranges from 15-28 kDa . This discrepancy between calculated and observed weights is important to note when analyzing experimental results. Researchers should validate their findings using appropriate positive controls, such as human skeletal muscle tissue or mouse ovary tissue, which have been confirmed to express detectable levels of COX7C .

How should sample preparation be optimized for COX7C detection in different tissues?

For optimal COX7C detection, tissue-specific sample preparation is crucial. In immunohistochemistry applications with paraffin-embedded tissues, antigen retrieval with TE buffer (pH 9.0) is suggested, though citrate buffer (pH 6.0) may serve as an alternative . For Western blot applications, positive signal detection has been validated in several tissue types, including mouse muscle, human fetal muscle, mouse heart, PC3 cells, mouse kidney, mouse small intestines, and 231 cells . When working with tissue lysates, loading approximately 40 μg of protein per lane in 12% SDS-PAGE gels has shown effective results .

What controls should be included when validating a new COX7C antibody?

When validating a new COX7C antibody, include the following controls:

• Positive tissue controls: Use human skeletal muscle, mouse ovary tissue, or MCF-7 cells, which have demonstrated positive expression .

• Negative controls: Omit primary antibody while maintaining all other steps.

• Knockdown/knockout validation: If available, use COX7C knockdown or knockout samples as specificity controls. Published studies have used this approach for validation .

• Cross-reactivity assessment: Test the antibody against related proteins, particularly other COX subunits, to ensure specificity.

• Molecular weight markers: Include reliable markers to confirm the observed molecular weight (15-28 kDa range) .

What secondary antibody options work best with rabbit polyclonal anti-COX7C antibodies?

For rabbit polyclonal anti-COX7C antibodies, the following secondary antibodies have been validated:

• Goat Anti-Rabbit IgG H&L Antibody (AP)

• Goat Anti-Rabbit IgG H&L Antibody (Biotin)

• Goat Anti-Rabbit IgG H&L Antibody (FITC)

• Goat Anti-Rabbit IgG H&L Antibody (HRP)

For Western blot applications, a dilution of 1/8000 for goat anti-rabbit IgG secondary antibody has shown effective results with 5-minute exposure times .

How can COX7C antibodies be utilized in neuronal research, particularly for studying mRNA transport?

Recent research has revealed that COX7C mRNA exhibits a unique transport mechanism in neurons, where it associates with and is co-transported with mitochondria along axons . To investigate this phenomenon:

• Combined antibody-mRNA detection approach: Perform single-molecule fluorescence in situ hybridization (smFISH) for COX7C mRNA together with immunostaining using COX7C antibodies and mitochondrial markers.

• Live imaging protocol: Use MS2-tagged COX7C mRNA constructs combined with mitochondrial staining for real-time visualization of co-transport.

• Comparative analysis: Compare COX7C with other mRNAs such as Cryab (crystallin B chain) that show less mitochondrial association to establish specificity of the transport mechanism .

Research has shown that up to 60% of COX7C mRNA colocalizes with mitochondria in primary motor neuron axons, significantly higher than its association with other axonal organelles like endosomes .

What methodologies combine COX7C antibodies with subcellular fractionation for studying mitochondrial protein import?

For studying COX7C's mitochondrial localization and import:

• Differential centrifugation protocol:

  • Homogenize cells in isotonic buffer

  • Separate nuclear, mitochondrial, and cytosolic fractions through sequential centrifugation steps

  • Verify fraction purity using organelle-specific markers

• Combined immunoblotting approach:

  • Use COX7C antibodies to detect the protein in different cellular fractions

  • Simultaneously probe for mitochondrial markers (e.g., VDAC) and cytosolic markers

  • Include markers for mitochondrially-encoded proteins like ND5 and COX1 as positive controls

• Cross-validation strategy:

  • Compare protein detection (via antibodies) with mRNA localization (via RT-qPCR or smFISH)

  • This approach has revealed that both COX7C protein and its corresponding mRNA show significant mitochondrial association

How can COX7C antibodies be applied in studies of diabetes-related sepsis biomarkers?

Research has identified COX7C as a potential biomarker for diabetes-related sepsis (DRS) . To apply COX7C antibodies in this context:

• Expression quantification methodology:

  • Use RT-qPCR to measure COX7C mRNA levels

  • Apply Western blot with COX7C antibodies for protein quantification

  • Calculate correlation coefficients between expression levels and disease severity

• Comparative biomarker panel approach:

  • Analyze COX7C alongside other identified biomarkers (UBE2D1, DLD, COX6C, ATP5C1, IDH1)

  • Establish relative expression patterns across normal and DRS groups

  • Perform correlation analysis between markers

• Statistical validation protocol:

  • Pearson's correlation analysis has shown strong relationships between COX7C expression and DRS (P<0.001, R=0.9034)

  • Use neural network prediction models to assess biomarker performance

  • Evaluate sensitivity and specificity through ROC curve analysis

How to address high background issues in COX7C immunohistochemistry?

High background in COX7C immunohistochemistry can be addressed through:

• Blocking optimization:

  • Extend blocking time to 1-2 hours

  • Test alternative blocking agents (BSA, normal serum, commercial blockers)

  • Include 0.1-0.3% Triton X-100 in blocking solution to reduce non-specific binding

• Antibody dilution adjustment:

  • For COX7C antibodies, the recommended IHC dilution range is 1:20-1:200

  • Prepare a dilution series to determine optimal concentration

  • Incubate at 4°C overnight rather than at room temperature

• Antigen retrieval modification:

  • For COX7C detection in paraffin-embedded tissues, TE buffer (pH 9.0) is recommended

  • Alternative protocol: citrate buffer (pH 6.0) with optimized heating time

  • Control retrieval duration carefully to prevent tissue damage

What strategies can resolve multiple bands in COX7C Western blots?

When encountering multiple bands in COX7C Western blots:

• Sample preparation refinement:

  • Use fresh samples and maintain cold temperatures during preparation

  • Add protease inhibitors to prevent degradation

  • Optimize lysis buffers for mitochondrial proteins

• Gel percentage adjustment:

  • Use 12-15% SDS-PAGE gels for better separation of the 7-28 kDa range

  • Validated protocols show successful detection using 12% SDS-PAGE

  • Run gels at lower voltage for improved resolution

• Antibody specificity verification:

  • Cross-reference with positive controls (human skeletal muscle, mouse ovary)

  • Test alternative antibody lots or sources

  • Consider that the observed molecular weight for COX7C typically ranges from 15-28 kDa, which may appear as multiple bands representing different post-translational modifications

How are COX7C antibodies being utilized in inflammation and COVID-19 research?

Recent studies have identified connections between COX7C and inflammatory responses, particularly in COVID-19:

• Pulmonary inflammation analysis:

  • Use COX7C antibodies to examine protein expression in lung tissue samples

  • Compare expression patterns between healthy controls and COVID-19 patients

  • Correlate with other respiratory chain components like NDUFA4

• Macrophage phenotyping protocol:

  • Apply COX7C antibodies in flow cytometry or immunofluorescence to characterize macrophage populations

  • Focus on FCN1+ and FCN1+SPP1+ macrophage clusters that predominate in severe COVID-19

  • Compare with S100A12+ and SPP1+ clusters found in other inflammatory conditions

• Mitochondrial remodeling assessment:

  • Examine changes in COX7C expression as part of cytochrome c oxidase remodeling during inflammation

  • Correlate with clinical parameters of disease severity

  • Integrate findings with transcriptomic analyses of inflammatory signatures

What methodological approaches can investigate the role of COX7C in neurodegenerative disorders?

To study COX7C in neurodegenerative contexts:

• Axonal transport visualization:

  • Combine COX7C antibodies with smFISH to visualize protein and mRNA localization

  • Use live imaging with MS2-tagged COX7C mRNA to track transport dynamics

  • Compare transport patterns between healthy neurons and disease models

• Mitochondrial function assessment:

  • Correlate COX7C expression with mitochondrial respiratory capacity

  • Measure oxygen consumption rates in normal vs. disease conditions

  • Analyze association between COX7C levels and mitochondrial membrane potential

• Translation regulation analysis:

  • Investigate the role of the mitochondrial targeting signal (MTS) in COX7C mRNA localization

  • Study how translation of the MTS affects mRNA transport and protein import

  • Examine the effects of MTS mutations on mRNA-mitochondria association