COX4I1 Monoclonal Antibody

What is COX4I1 and what is its function in cellular metabolism?

COX4I1 is a nuclear-encoded subunit of cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain. It couples the transfer of electrons from cytochrome c to molecular oxygen and contributes to the proton electrochemical gradient across the inner mitochondrial membrane. The complex consists of 13 mitochondrial- and nuclear-encoded subunits, with the mitochondrially-encoded subunits performing electron transfer and proton pumping activities . COX4I1 is one of the nuclear-encoded subunits, though its precise regulatory function remains under investigation, with current research suggesting it plays a role in the regulation and assembly of the complex .

How does COX4I1 differ from other cytochrome c oxidase subunits?

COX4I1 belongs to the cytochrome c oxidase IV family and is specifically the nuclear-encoded subunit IV isoform 1 of the mitochondrial respiratory chain enzyme. Unlike other COX subunits, COX4I1 is ubiquitously expressed across tissues, while its counterpart isoform 2 (COX4I2) shows tissue-specific expression, primarily in lung tissue . The gene encoding COX4I1 is located at the 3' end of the NOC4 (neighbor of COX4) gene in a head-to-head orientation, and interestingly, it shares a promoter with this gene . With a calculated molecular weight of 19.6 kDa (though typically observed at 17-18 kDa in experimental conditions), COX4I1 serves as one of the key structural and regulatory components of the COX complex .

What makes COX4I1 a reliable mitochondrial marker in experimental research?

COX4I1 has become a standard mitochondrial marker in experimental research due to several key attributes. First, its consistent and ubiquitous expression across diverse tissue types ensures reliable detection in various experimental systems . Unlike some mitochondrial proteins that might show variable expression or isoform switching under different conditions, COX4I1 maintains relatively stable expression, making it suitable as a loading control for mitochondrial content normalization . Furthermore, its exclusive localization to mitochondria without significant presence in other subcellular compartments makes it a specific marker for mitochondrial isolation and purity assessment . Recent studies have also validated its utility through multiple detection methods including Western blot, immunohistochemistry, and immunofluorescence with consistent results across these platforms .

What are the validated applications for COX4I1 monoclonal antibodies?

COX4I1 monoclonal antibodies have been extensively validated for multiple research applications. Western blotting (WB) is the most common application, with antibodies typically functioning at dilutions ranging from 1:500-1:50000 depending on the specific antibody clone . Immunohistochemistry (IHC) applications using COX4I1 antibodies have been validated on various tissue types, including human colon cancer, lung cancer, prostate cancer, pancreas, and heart tissues, generally at dilutions of 1:250-1:10000 . Immunofluorescence (IF) and immunocytochemistry (ICC) applications typically require dilutions of 1:50-1:200 . Flow cytometry has also been validated, particularly useful for assessing mitochondrial content in individual cells . Some antibodies have additionally been tested for immunoprecipitation (IP) applications, allowing researchers to isolate COX4I1-containing protein complexes .

How should sample preparation be optimized for detection of COX4I1 using monoclonal antibodies?

Optimal sample preparation for COX4I1 detection varies by application but follows some general principles. For Western blotting, complete lysis buffers containing mild detergents (such as Triton X-100 or NP-40) are recommended to maintain protein structure while releasing mitochondrial proteins . For tissue samples undergoing immunohistochemistry, heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has shown excellent results, though citrate buffer (pH 6.0) has also been effectively used as an alternative . For immunofluorescence applications on cultured cells, fixation with 4% paraformaldehyde followed by permeabilization with Triton X-100 provides optimal results, preserving mitochondrial morphology while allowing antibody access . Flow cytometry applications require careful permeabilization of fixed cells to maintain mitochondrial integrity while allowing antibody penetration; 4% paraformaldehyde fixation followed by permeabilization buffer treatment has been validated for this purpose .

What dilution ranges are most effective for different experimental applications?

Optimal dilution ranges vary significantly by both application type and specific antibody clone. For Western blotting applications, most COX4I1 monoclonal antibodies work effectively at dilutions between 1:500-1:50000, with higher-quality antibody preparations (like those designated "Picoband®") generally allowing for more dilute applications . For immunohistochemistry, recommended dilutions typically range from 1:250-1:10000, with 1:1000-1:2500 being a common starting point for optimization . Immunofluorescence and immunocytochemistry applications generally require more concentrated antibody solutions, with typical dilutions ranging from 1:50-1:200 . Flow cytometry applications using COX4I1 antibodies generally use concentrations around 1μg per 10^6 cells . It's important to note that these ranges are starting points, and researchers should perform dilution series optimization for their specific experimental systems and antibody lots to determine optimal signal-to-noise ratios.

What controls should be included when using COX4I1 antibodies in research applications?

Rigorous experimental design with appropriate controls is essential when working with COX4I1 antibodies. Positive controls should include samples known to express COX4I1, such as HeLa, HEK-293, and HepG2 cells, which have been validated to show strong and consistent COX4I1 expression . Negative controls should include samples where primary antibody is omitted but secondary antibody is applied, to assess non-specific binding of the detection system . For more advanced validation, RNAi knockdown of COX4I1 provides a powerful negative control, as demonstrated in enhanced validation protocols by several manufacturers . Isotype controls using non-specific mouse IgG (matching the isotype of the COX4I1 antibody being used, typically IgG1 or IgG2a) at equivalent concentrations help evaluate potential non-specific binding due to Fc receptor interactions or other non-target-specific mechanisms . For Western blotting applications, molecular weight markers should always be included to confirm the detection of bands at the expected size (typically observed at 17-18 kDa for COX4I1) .

How can researchers validate the specificity of a COX4I1 monoclonal antibody?

Validating antibody specificity is crucial for ensuring reliable research results. Several complementary approaches are recommended. First, Western blot analysis should confirm a single band at the expected molecular weight (17-18 kDa for COX4I1) . Multiple cell lines or tissue types should be tested to ensure consistent detection patterns, as exemplified in validation studies showing COX4I1 detection across human cell lines including HEPG2, A549, HEK293, T47D, CACO-2, K562, and HeLa . Genetic approaches provide the most stringent validation - using siRNA or CRISPR/Cas9 to knock down or knock out COX4I1 expression should result in corresponding reduction or elimination of the antibody signal . Peptide competition assays, where the antibody is pre-incubated with purified COX4I1 peptide (corresponding to the immunogen sequence) before application to samples, should eliminate specific binding if the antibody is truly target-specific . Cross-reactivity testing with related proteins, particularly COX4I2 (the lung-specific isoform), should be performed to ensure the antibody specifically recognizes only COX4I1 .

What are common technical issues when using COX4I1 antibodies and how can they be resolved?

Several technical challenges can arise when working with COX4I1 antibodies. One common issue is weak or absent signal in Western blotting, which may be addressed by optimizing protein extraction methods specifically for mitochondrial proteins, using mitochondrial enrichment protocols, or adjusting sample heating conditions to prevent aggregation of membrane proteins . High background in immunohistochemistry applications can be minimized by optimizing blocking conditions (using 10% goat serum has been validated in multiple protocols) and carefully titrating both primary and secondary antibody concentrations . For immunofluorescence applications, autofluorescence from mitochondria, particularly in fixed tissues, can interfere with specific signal detection; this can be mitigated by using appropriate quenching procedures or spectral unmixing during imaging . Cross-reactivity with COX4I2 has been reported with some antibodies; researchers should specifically select antibodies verified not to cross-react with this isoform, particularly when studying lung tissues where both isoforms may be expressed .

How is COX4I1 implicated in cancer research and what methodological approaches are relevant?

Recent research has revealed significant roles for COX4I1 in cancer biology, with important methodological implications. A groundbreaking 2025 study identified COX4I1 as a novel vulnerability in acute myeloid leukemia (AML) through a cell signaling-focused CRISPR screen . This research demonstrated that depletion of COX4I1 hindered leukemia cell proliferation and impacted in vivo AML progression by inducing mitochondrial stress and ferroptosis . Methodologically, researchers investigating COX4I1 in cancer contexts should consider several approaches: immunohistochemical analysis of COX4I1 expression in cancer tissues compared to normal counterparts (protocols using 1μg/ml antibody concentration with overnight 4°C incubation have been validated) ; assessment of mitochondrial respiratory capacity in cancer cells following COX4I1 manipulation using techniques like Seahorse analysis; and combining COX4I1 targeting with established cancer therapeutics such as venetoclax, which has demonstrated synergistic effects in AML models . CRISPR gene tiling scans, coupled with mitochondrial proteomics, have also been effective in dissecting critical regions within COX4I1 essential for leukemia cell survival .

How can COX4I1 antibodies be utilized in mitochondrial biogenesis and dynamics studies?

COX4I1 antibodies serve as valuable tools for investigating mitochondrial biogenesis and dynamics. For biogenesis studies, quantitative Western blotting with COX4I1 antibodies (typically at 1:5000-1:50000 dilutions) provides a reliable measure of mitochondrial content changes in response to various stimuli or genetic manipulations . Immunofluorescence microscopy using COX4I1 antibodies allows visualization of mitochondrial network morphology and distribution, particularly useful when studying mitochondrial fission/fusion dynamics or mitophagy processes . For more sophisticated analyses, combining COX4I1 immunostaining with other mitochondrial markers that localize to different subcompartments (matrix, inner membrane, outer membrane) can reveal the spatial organization of mitochondrial components during dynamic processes . Flow cytometry with COX4I1 antibodies (using protocols with 4% paraformaldehyde fixation and appropriate permeabilization) enables quantitative assessment of mitochondrial content at the single-cell level, allowing researchers to identify heterogeneous responses within cell populations . Time-course experiments tracking COX4I1 expression following induction of mitochondrial biogenesis (e.g., with exercise, caloric restriction, or pharmacological activators) have been particularly informative in understanding the temporal regulation of mitochondrial expansion .

What are the critical quality attributes to evaluate when selecting a COX4I1 monoclonal antibody?

When selecting a COX4I1 monoclonal antibody, researchers should evaluate several critical quality attributes to ensure reliable experimental outcomes. Clone specificity should be assessed through validation data showing detection of a single band at the expected molecular weight (17-18 kDa) in Western blot applications across multiple cell lines . Cross-reactivity profiles should be examined, particularly regarding other COX isoforms; high-quality antibodies will specifically detect COX4I1 without recognizing COX4I2 . The antibody's validation across multiple applications is important - comprehensive validation data should show successful use in Western blotting, immunohistochemistry, immunofluorescence, and potentially flow cytometry or immunoprecipitation, depending on the researcher's needs . Enhanced validation methods, such as RNAi knockdown confirmation of specificity, provide additional confidence in antibody performance . Form and storage stability information is also crucial - most COX4I1 antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, and are stable for one year when stored at -20°C .

How can researchers interpret validation images for COX4I1 antibodies to assess their suitability?

Validation images provide crucial information for assessing antibody performance in specific applications. For Western blot validation images, researchers should look for a single, clear band at 17-18 kDa across multiple cell lines or tissue lysates (such as HEPG2, A549, HEK293, T47D, CACO-2, K562, and HeLa cells, which have been validated to express COX4I1) . Band intensity should correlate with expected mitochondrial content across different samples. For immunohistochemistry validation images, researchers should assess staining patterns showing the expected mitochondrial distribution within cells, typically appearing as cytoplasmic granular staining consistent with mitochondrial localization . Background staining should be minimal, and negative control sections (without primary antibody) should show no signal. Immunofluorescence validation images should demonstrate co-localization with other established mitochondrial markers and show the characteristic mitochondrial network morphology . Flow cytometry validation histograms should show clear separation between the negative control (isotype control antibody or unstained cells) and the COX4I1-stained population .

What immunogen strategies yield the most specific COX4I1 monoclonal antibodies?

The choice of immunogen strategy significantly impacts the specificity and utility of resulting COX4I1 monoclonal antibodies. Based on the available data, several approaches have proven successful. Fusion protein approaches using the full-length COX4I1 protein have generated highly specific antibodies, as exemplified by antibodies developed using the COX4I1 fusion protein Ag20551 . Synthetic peptide immunogens corresponding to specific regions of the COX4I1 protein sequence (such as "LATRVFSLVGKRAISTSVCVRAHESVVKSEDFSLPAYMDRRDHPLPEVAHVKHLSASQKALKEKEKASWSSLSMDEKVELYRIKFKESFAEMNRGSNEWKT") have also yielded high-quality antibodies . For developing isoform-specific antibodies that distinguish between COX4I1 and COX4I2, immunogens derived from regions showing the greatest sequence divergence between these isoforms have been most successful . Production of monoclonal antibodies (rather than polyclonal) provides greater consistency and specificity, with mouse hosts (particularly generating IgG1 or IgG2a isotypes) being the predominant system for COX4I1 antibody development .

How is COX4I1 research contributing to understanding mitochondrial quality control mechanisms?

COX4I1 has emerged as a critical component in understanding mitochondrial quality control mechanisms. Recent studies have demonstrated that depletion of COX4I1 induces mitochondrial stress and ferroptosis, disrupting mitochondrial ultrastructure and oxidative phosphorylation . This finding positions COX4I1 as a potential checkpoint in mitochondrial homeostasis pathways. Methodologically, researchers investigating these processes should employ complementary approaches: electron microscopy to visualize changes in mitochondrial morphology following COX4I1 manipulation; measurements of mitochondrial reactive oxygen species production using fluorescent indicators; membrane potential assessment using potentiometric dyes; and gene expression analysis focusing on mitochondrial unfolded protein response markers . The interaction between COX4I1 and mitochondrial quality control pathways like PINK1/Parkin-mediated mitophagy represents an exciting frontier, with immunoprecipitation studies using COX4I1 antibodies potentially revealing novel protein-protein interactions involved in recognizing and removing damaged mitochondria .

What new insights have been gained about the role of COX4I1 in therapeutic resistance mechanisms?

Recent research has uncovered significant connections between COX4I1 and therapeutic resistance mechanisms, particularly in cancer contexts. A groundbreaking 2025 study revealed that COX4I1 depletion or pharmacological inhibition of Complex IV (using chlorpromazine) synergized with venetoclax, a BCL-2 inhibitor used in leukemia treatment . This finding suggests that mitochondrial respiratory function, regulated in part by COX4I1, plays a critical role in resistance to apoptosis-inducing therapies. For researchers investigating these mechanisms, several methodological approaches are valuable: combination treatment studies evaluating synergy between mitochondrial-targeting compounds and standard therapeutics; time-course analyses of COX4I1 expression in treatment-resistant versus sensitive cells using quantitative Western blotting or immunofluorescence; and functional respirometry to assess whether changes in oxidative phosphorylation correlate with treatment response . The mechanistic link between mitochondrial respiratory function and drug resistance pathways represents a promising avenue for developing strategies to overcome therapeutic resistance in various diseases.

How are advanced imaging techniques enhancing COX4I1 research?

Advanced imaging techniques are revolutionizing COX4I1 research by providing unprecedented insights into its dynamics and interactions. Super-resolution microscopy (such as STED, STORM, or PALM) using COX4I1 antibodies allows visualization of its precise localization within mitochondrial cristae structures, going beyond the diffraction limit of conventional microscopy . Live-cell imaging approaches, particularly when combined with genetically encoded COX4I1 fusion proteins, enable real-time tracking of mitochondrial dynamics in response to various stimuli . Correlative light and electron microscopy (CLEM) combines the specificity of fluorescent COX4I1 labeling with the ultrastructural detail of electron microscopy, providing contextual information about COX4I1 localization relative to mitochondrial substructures . Multiplexed imaging approaches, simultaneously detecting COX4I1 alongside other mitochondrial proteins, metabolic sensors, or cell death markers, reveal the complex relationships between mitochondrial respiratory function and broader cellular processes . Quantitative image analysis algorithms now allow automated assessment of mitochondrial morphology, network connectivity, and COX4I1 distribution across large datasets, enabling high-throughput screening approaches previously impossible with manual analysis .