COX7B Antibody

What is COX7B and what is its biological significance?

COX7B (cytochrome c oxidase subunit VIIb) is an 80 amino acid protein (9 kDa) that belongs to the cytochrome c oxidase VIIb family . It functions as one of the nuclear-coded polypeptide chains of cytochrome c oxidase, the terminal oxidase in mitochondrial electron transport . COX7B is a critical component of the respiratory chain that catalyzes the reduction of oxygen to water . The protein plays a significant role in proper central nervous system development in vertebrates . As part of complex IV of the mitochondrial electron transport chain, COX7B is integral to oxidative phosphorylation processes that generate cellular energy, making it essential for normal cellular metabolism and function .

How is COX7B expression regulated in normal versus disease states?

COX7B expression varies significantly between normal and disease states, particularly in cancer. Analysis of The Cancer Genome Atlas (TCGA) data reveals that COX7B is overexpressed in multiple cancer types including breast cancer (BRCA), cervical cancer (CESC), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and uterine corpus endometrial carcinoma (UCEC) . Conversely, COX7B shows reduced expression in kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), rectal adenocarcinoma (READ), and thyroid carcinoma (THCA) . In esophageal carcinoma specifically, COX7B expression is significantly higher in tumor tissues compared to non-tumor tissues, with elevated expression particularly notable in advanced disease stages (stage IV compared to stage I) .

What is the structural and functional relationship between COX7B and other components of the electron transport chain?

COX7B functions as a component of cytochrome c oxidase (complex IV), the terminal enzyme in the mitochondrial respiratory chain . This complex works cooperatively with other multisubunit complexes including succinate dehydrogenase (complex II) and ubiquinol-cytochrome c oxidoreductase (complex III) to transfer electrons derived from NADH and succinate to molecular oxygen . Within this system, COX7B contributes to the creation of an electrochemical gradient across the inner mitochondrial membrane that drives ATP production . The enzyme catalyzes the reduction of oxygen to water through a process where electrons from reduced cytochrome c in the intermembrane space are transferred via copper centers and heme groups to the active site, which then reduces molecular oxygen using electrons from cytochrome c and protons from the mitochondrial matrix .

What criteria should researchers consider when selecting the appropriate COX7B antibody for their experiments?

When selecting a COX7B antibody, researchers should consider several critical factors to ensure experimental success. First, verify the antibody's reactivity with your species of interest; available antibodies show reactivity with human, mouse, and rat samples . Second, confirm the antibody's validated applications (Western Blot, IHC, ELISA, ICC/IF) align with your experimental needs . Third, consider antibody type (polyclonal vs. monoclonal) based on your specific requirements—polyclonal antibodies like 11417-2-AP offer broad epitope recognition, while monoclonal antibodies like EPR9326(B) provide higher specificity . Fourth, examine validation data, including knockout cell line testing, as demonstrated with ab140629 which showed specific reactivity with COX7B in wild-type HeLa cells and loss of signal in knockout cells . Finally, consider the antibody's immunogen and how it relates to your research question—for instance, whether you need an antibody that recognizes specific domains or post-translational modifications of COX7B.

How can researchers validate the specificity of a COX7B antibody for their experimental system?

Validating COX7B antibody specificity requires a multi-faceted approach. Begin with Western blot analysis using positive control samples known to express COX7B, such as mouse brain tissue, MCF-7 cells, or rat brain tissue, verifying the detection of a band at the expected molecular weight of 9 kDa . Include a negative control via COX7B knockdown or knockout cells, as demonstrated with the ab140629 antibody tested against wild-type and COX7B knockout HeLa cells . For immunohistochemistry applications, compare staining patterns in tissues known to express COX7B (like mouse heart tissue) with appropriate negative controls . Peptide competition assays can further confirm specificity by pre-incubating the antibody with the immunizing peptide to block specific binding. Additionally, cross-validate results using multiple antibodies targeting different epitopes of COX7B or orthogonal detection methods such as mass spectrometry. Finally, verify antibody performance across different experimental conditions by testing various fixation methods, antigen retrieval protocols, and dilutions to optimize signal-to-noise ratio.

What are the optimal dilution ranges for COX7B antibodies in different experimental applications?

The optimal dilution ranges for COX7B antibodies vary significantly depending on the specific application and the antibody being used. For Western blot applications using the 11417-2-AP antibody, the recommended dilution range is 1:2000-1:10000 . This wide range suggests that researchers should begin with a middle dilution (e.g., 1:5000) and adjust based on signal intensity and background levels. For immunohistochemistry (IHC) applications with the same antibody, a dilution range of 1:50-1:500 is recommended . The significant difference between WB and IHC dilutions reflects the different protein concentrations and detection sensitivities of these methods. It's important to note that optimal dilutions may be sample-dependent, requiring titration in each specific testing system to obtain optimal results . When using different antibodies such as the EPR9326(B) monoclonal antibody (ab140629), researchers should consult specific product documentation for recommended dilutions in applications including IHC-P, WB, and ICC/IF . As a general practice, researchers should always perform dilution series experiments to determine the optimal antibody concentration for their specific experimental conditions, sample types, and detection methods.

What are the optimal protocols for using COX7B antibodies in Western blot applications?

For optimal Western blot detection of COX7B using antibodies such as 11417-2-AP, researchers should follow this methodological approach: First, prepare protein samples from appropriate tissues (mouse/rat brain or MCF-7 cells have shown good results) . Use standard SDS-PAGE with appropriate percentage gels (15-18% recommended for the small 9 kDa COX7B protein) . After electrophoresis, transfer proteins to a PVDF or nitrocellulose membrane using a wet transfer system with methanol-containing buffer for efficient transfer of small proteins. Block the membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature. Apply the primary COX7B antibody at the recommended dilution (1:2000-1:10000 for 11417-2-AP) , incubating overnight at 4°C. After washing with TBST (3-5 times, 5 minutes each), apply an appropriate HRP-conjugated secondary antibody (anti-rabbit for most available COX7B antibodies) at 1:5000-1:10000 dilution for 1 hour at room temperature. Perform final washes and develop using enhanced chemiluminescence. When analyzing results, expect to observe a specific band at approximately 9 kDa, consistent with the calculated molecular weight of COX7B . Include appropriate positive controls (known COX7B-expressing samples) and loading controls (such as GAPDH) to ensure result validity and reliability.

What special considerations should be taken for immunohistochemical detection of COX7B?

Immunohistochemical detection of COX7B requires several special considerations for optimal results. First, antigen retrieval is critical—for the 11417-2-AP antibody, the suggested method is using TE buffer at pH 9.0, although citrate buffer at pH 6.0 may serve as an alternative . This step is essential due to the mitochondrial localization of COX7B, which may require more aggressive retrieval methods to expose epitopes. Second, researchers should optimize antibody dilution within the recommended range (1:50-1:500) , starting with a middle dilution and adjusting based on signal strength and background levels. Third, selection of appropriate tissue controls is important—mouse heart tissue has been validated for positive IHC detection with COX7B antibodies . Fourth, researchers should consider double immunostaining with mitochondrial markers to confirm the subcellular localization pattern. Fifth, blocking endogenous peroxidase activity and using appropriate blocking sera is essential to reduce background staining. Sixth, signal amplification systems may be beneficial for detecting low-abundance targets like COX7B. Finally, careful optimization of incubation times, temperatures, and washing steps is necessary to achieve the optimal signal-to-noise ratio. Researchers should also consider sample preparation variables, including fixation method and duration, which can significantly impact epitope preservation and antibody accessibility.

How can researchers effectively use COX7B antibodies for protein co-localization studies?

For effective protein co-localization studies using COX7B antibodies, researchers should implement a comprehensive methodological approach. Begin by selecting compatible primary antibodies that can be distinguished by species or isotype to allow for differential secondary antibody labeling—for example, pairing a rabbit anti-COX7B antibody with mouse antibodies against other mitochondrial proteins or potential interacting partners . For immunofluorescence applications, use fluorescently-labeled secondary antibodies with well-separated emission spectra (e.g., Alexa Fluor 488 and 594) to minimize bleed-through. Include essential controls: single-labeled samples to establish detection thresholds and rule out cross-reactivity, and negative controls omitting primary antibodies to assess nonspecific binding. When imaging, use confocal microscopy for optimal spatial resolution, particularly important for mitochondrial proteins like COX7B where precise subcellular localization is critical. Z-stack imaging can provide three-dimensional information about co-localization patterns. For quantitative co-localization analysis, employ specialized software (e.g., ImageJ with Coloc2 plugin) to calculate parameters such as Pearson's correlation coefficient, Mander's overlap coefficient, or intensity correlation quotient. Given COX7B's function in the mitochondrial respiratory chain , co-localization studies with other components of complex IV or other mitochondrial markers (like TOMM20 or MitoTracker dyes) can provide valuable insights into its functional integration within the mitochondrial network.

What is the relationship between COX7B expression and immune cell infiltration in tumor microenvironments?

Research has uncovered significant relationships between COX7B expression and immune cell infiltration in tumor microenvironments, particularly in esophageal carcinoma (ESCA). Spearman correlation analysis revealed that COX7B expression is inversely related to several important immune cell populations, specifically T follicular helper cells (TFH), central memory T cells (Tcm), natural killer (NK) cells, and mast cells . This negative correlation suggests that increased COX7B expression may contribute to an immunosuppressive tumor microenvironment, potentially facilitating tumor immune evasion. The relationship with NK cells is particularly noteworthy, as these cells play critical roles in cancer immunosurveillance. The inverse correlation between COX7B and mast cells adds complexity to our understanding, as mast cells can have both pro- and anti-tumorigenic functions depending on the tumor context. This immune cell infiltration pattern aligns with previous research showing that immune-inhibited cell types (such as reduced CD8+ T cells and increased M2 macrophages) are often present in high numbers in ESCA . These findings suggest that COX7B may influence the composition of the tumor immune microenvironment, potentially affecting patient response to immunotherapies. Given the growing importance of immune checkpoint inhibitors in cancer treatment, including for ESCA, the relationship between COX7B and immune cell populations could have significant implications for predicting immunotherapy response and developing combination treatment strategies.

What molecular pathways and functions are associated with COX7B in cancer and other diseases?

Gene Set Enrichment Analysis (GSEA) and related pathway analyses have identified several key molecular pathways and functions associated with COX7B in disease contexts. In esophageal carcinoma, COX7B has been linked to cytokine-receptor interaction, extracellular matrix receptor interaction, focal adhesion, JAK/STAT signaling pathway, and neuroactive ligand-receptor interaction . These pathways suggest that beyond its canonical role in mitochondrial function, COX7B may influence tumor progression through effects on cell adhesion, migration, and signaling networks. The association with JAK/STAT signaling is particularly significant given this pathway's established role in cancer cell proliferation, survival, and immune evasion. At the functional level, COX7B serves as a component of cytochrome c oxidase (complex IV), the terminal enzyme in the mitochondrial electron transport chain driving oxidative phosphorylation . This respiratory chain complex works cooperatively with other multisubunit complexes to transfer electrons derived from NADH and succinate to molecular oxygen, creating an electrochemical gradient that drives ATP synthesis . Beyond cancer, COX7B plays a crucial role in proper central nervous system development in vertebrates , suggesting its dysregulation could contribute to neurological disorders. The protein has also been implicated in breast cancer brain tropism, indicating a potential role in determining cancer metastatic patterns . These diverse functions highlight COX7B as a multifaceted protein with implications for various disease processes beyond its primary mitochondrial role.

What are the challenges in detecting post-translational modifications of COX7B?

Detecting post-translational modifications (PTMs) of COX7B presents several significant challenges due to both technical limitations and the protein's inherent characteristics. First, COX7B's small size (80 amino acids, 9 kDa) provides limited potential modification sites and creates detection difficulties, as modified peptides may be overlooked in standard proteomic workflows. Second, COX7B's mitochondrial localization presents extraction challenges, requiring specialized protocols to maintain the integrity of labile PTMs during isolation. Third, there is a current lack of commercially available modification-specific antibodies for COX7B, necessitating the use of general PTM antibodies coupled with COX7B immunoprecipitation, which may have sensitivity limitations. Fourth, the low abundance of modified forms relative to unmodified COX7B requires enrichment strategies such as affinity chromatography or specific precipitation methods. Fifth, certain modifications may alter antibody recognition sites, potentially causing false negatives with antibodies raised against unmodified sequences. Sixth, PTM crosstalk (where one modification influences the presence of others) necessitates simultaneous detection of multiple modifications, requiring sophisticated mass spectrometry approaches. To address these challenges, researchers should consider employing targeted mass spectrometry approaches such as parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM), using metabolic labeling with modification-specific precursors, and developing proximity ligation assays to detect specific modified forms of COX7B in situ.

What strategies can overcome challenges in co-immunoprecipitation of COX7B with interacting partners?

Successfully co-immunoprecipitating COX7B with its interacting partners requires specialized strategies to overcome inherent challenges. First, preserve protein complexes by using gentle lysis buffers containing digitonin or n-dodecyl-β-D-maltoside rather than harsh detergents like SDS, which are particularly important for maintaining mitochondrial membrane protein associations . Second, consider crosslinking approaches (using formaldehyde or specialized crosslinkers) to stabilize transient interactions before cell lysis, especially important for capturing dynamic respiratory chain complex associations. Third, optimize antibody selection by testing multiple COX7B antibodies with different epitopes (such as 11417-2-AP or EPR9326(B)) to identify those that don't interfere with protein-protein interaction regions. Fourth, employ a sequential immunoprecipitation approach (tandem IP) to increase specificity when investigating complex multi-protein assemblies. Fifth, validate interactions through reciprocal co-IPs using antibodies against suspected interacting partners and confirming COX7B presence. Sixth, use proximity-dependent labeling methods like BioID or APEX2 by fusing these enzymes to COX7B to identify proximal proteins in the native cellular environment. Seventh, consider size exclusion chromatography coupled with blue native PAGE before immunoprecipitation to isolate intact respiratory chain complexes containing COX7B. Finally, complement co-IP results with orthogonal approaches such as fluorescence resonance energy transfer (FRET), proximity ligation assays (PLA), or hydrogen-deuterium exchange mass spectrometry (HDX-MS) to strengthen confidence in identified protein-protein interactions.

How can COX7B antibodies be utilized in developing new diagnostic approaches for cancer?

COX7B antibodies offer promising potential for developing novel cancer diagnostic approaches based on recent research findings. First, immunohistochemical detection platforms could be developed for tissue biopsies, leveraging the differential expression of COX7B between tumor and normal tissues across multiple cancer types, with particular relevance for esophageal carcinoma where COX7B overexpression correlates with advanced disease stage . Second, liquid biopsy applications could be explored by developing assays to detect COX7B in circulating tumor cells or extracellular vesicles using antibody-based capture and detection systems. Third, multiplexed immunoassays combining COX7B antibodies with other cancer biomarkers could enhance diagnostic accuracy—particularly valuable given COX7B's prognostic value in ESCA with an AUC of 0.788 . Fourth, antibody-based imaging approaches using labeled COX7B antibodies could potentially enable non-invasive visualization of tumors with high COX7B expression. Fifth, researchers could develop COX7B-targeted companion diagnostics to identify patients who might benefit from therapies affecting mitochondrial function or the JAK/STAT pathway, which has been associated with COX7B function . Sixth, incorporating COX7B into multiparameter prognostic nomograms (as demonstrated for ESCA) could improve risk stratification, helping clinicians make more informed treatment decisions. To validate these approaches, researchers should conduct large-scale studies across diverse patient populations, comparing COX7B-based diagnostics with current gold standards while assessing sensitivity, specificity, and predictive values in both primary diagnosis and recurrence monitoring scenarios.

What are the emerging applications of COX7B antibodies in studying mitochondrial dynamics and bioenergetics?

COX7B antibodies are enabling innovative research approaches in mitochondrial biology beyond their traditional detection applications. Researchers are now employing these antibodies in advanced live-cell imaging techniques by generating cell lines expressing COX7B fusion proteins (with fluorescent tags or self-labeling enzyme tags like SNAP or Halo) and validating these models against endogenous COX7B antibody staining patterns . This allows real-time visualization of complex IV dynamics. Super-resolution microscopy (STORM, PALM, or STED) using COX7B antibodies is revealing the precise nanoscale organization of respiratory complexes within cristae, advancing our understanding of mitochondrial ultrastructure. Proximity labeling approaches coupling COX7B antibodies with peroxidase-mediated biotinylation are mapping the mitochondrial interactome in various physiological and pathological states. Researchers are developing antibody-based sensors by conjugating COX7B antibodies with environment-sensitive fluorophores that respond to changes in mitochondrial membrane potential or local pH, creating new tools for monitoring bioenergetic states. Correlative light and electron microscopy (CLEM) using COX7B antibodies is linking functional data from fluorescence imaging with ultrastructural information. Single-molecule tracking of COX7B is revealing the mobility and turnover of respiratory complexes within mitochondrial membranes. Finally, researchers are exploring antibody-based detection of COX7B in isolated mitochondrial subpopulations to investigate the heterogeneity of mitochondrial function within cells and tissues. These emerging applications are expanding the utility of COX7B antibodies beyond conventional protein detection to address fundamental questions in mitochondrial biology.

How might COX7B serve as a therapeutic target, and what role could COX7B antibodies play in drug development?

COX7B presents several promising avenues as a therapeutic target, with COX7B antibodies potentially playing crucial roles in drug development processes. First, as COX7B is overexpressed in multiple cancer types and correlates with poor prognosis in esophageal carcinoma , developing therapeutic antibodies or antibody-drug conjugates targeting COX7B-expressing cancer cells could represent a novel treatment approach. Second, given the inverse relationship between COX7B expression and immune cell infiltration (particularly TFH, Tcm, NK cells, and mast cells) , combination therapies targeting COX7B alongside immune checkpoint inhibitors might enhance immunotherapy efficacy by modulating the tumor microenvironment. Third, COX7B antibodies could serve as valuable tools in drug screening assays to identify small molecules that modulate COX7B expression or function, which would be particularly relevant for cancers where COX7B overexpression drives disease progression. Fourth, the association between COX7B and the JAK/STAT signaling pathway suggests potential synergies between COX7B-targeted therapies and existing JAK inhibitors. Fifth, researchers could develop bifunctional antibodies targeting both COX7B and immune effector cells to redirect immune responses against COX7B-expressing tumors. In the drug development process, COX7B antibodies would be essential for target validation, pharmacodynamic biomarker development, and patient stratification in clinical trials. As research progresses, investigating how COX7B modulation affects mitochondrial function in different tissues will be crucial to understand and mitigate potential off-target effects of COX7B-directed therapies.