NDUFAF1 Antibody

What is NDUFAF1 and what is its function in mitochondrial biology?

NDUFAF1 (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, assembly factor 1) is a critical assembly factor for mitochondrial complex I. It is the human homolog of Neurospora crassa CIA30 protein . As part of the MCIA (mitochondrial complex I assembly) complex, NDUFAF1 plays an essential role in the regulation and assembly/stability of human complex I, which is the largest multiprotein enzyme of the oxidative phosphorylation system .

Research demonstrates that knockdown of NDUFAF1 leads to reduced amount and activity of complex I . Mutations in the NDUFAF1 gene are associated with mitochondrial complex I deficiency, which can result in severe mitochondrial disease . The protein has a calculated molecular weight of 38 kDa but is typically observed at approximately 32-33 kDa in Western blot analyses .

What are the validated applications for NDUFAF1 antibodies in research protocols?

Based on peer-reviewed literature and validated protocols, NDUFAF1 antibodies have been successfully employed in the following applications:

For Western blotting, NDUFAF1 antibodies have been used to detect the protein in various cell lines including HEK-293, Jurkat, HeLa, and human heart tissue . For immunohistochemistry, protocols typically recommend antigen retrieval with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0 .

How can researchers verify the specificity of NDUFAF1 antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For NDUFAF1 antibodies, researchers should implement the following approaches:

First, confirm that the detected band appears at the expected molecular weight. While the calculated molecular weight of NDUFAF1 is 38 kDa, it is typically observed at approximately 32-33 kDa in Western blots . This discrepancy should be acknowledged when interpreting results.

Second, implement genetic knockdown controls. siRNA-mediated knockdown of NDUFAF1 should result in diminished signal intensity, as demonstrated in multiple studies investigating complex I assembly . This approach not only validates antibody specificity but can also be used to study the functional consequences of NDUFAF1 reduction.

Third, consider using multiple antibodies raised against different epitopes of NDUFAF1. The search results indicate several validated antibodies are available, including polyclonal (e.g., 15181-1-AP) and monoclonal (e.g., EPR2796, EPR2795) options .

Fourth, when working with non-human models, verify cross-reactivity. Available data indicates sequence homology with mouse (82%) and rat (84%) orthologs , but experimental validation is still recommended.

How can NDUFAF1 antibodies be utilized to investigate complex I assembly intermediates?

NDUFAF1 antibodies provide powerful tools for dissecting the complex I assembly pathway through several sophisticated approaches:

Blue Native PAGE (BN-PAGE) coupled with immunoblotting allows identification of NDUFAF1-containing assembly intermediates. Research has demonstrated that NDUFAF1 is associated with two complexes of approximately 600 and 700 kDa . The relative distribution of these complexes is altered in patients with complex I deficiency, making this approach valuable for both research and potential diagnostic applications.

For temporal analysis of complex I assembly, pulse-chase labeling experiments combined with immunoprecipitation using NDUFAF1 antibodies have revealed critical insights. Studies show strong interaction of NDUFAF1 with newly synthesized mtDNA-encoded subunits, particularly ND2 and to a lesser extent with ND1, ND3, and ND4L immediately after pulse labeling . These interactions decrease over time (3 hours post-chase) and are virtually absent after 24 hours when subunits have assembled into complex I . This methodology demonstrates how NDUFAF1 antibodies can track the dynamic process of complex assembly.

Recent research utilizing cryo-electron microscopy has determined structures of assembly intermediates associated with NDUFAF1, revealing that subunits ND2 and NDUFC2 together with NDUFAF1 form the nucleation point of the assembly pathway . Such studies employed NDUFAF1 antibodies for initial identification and purification of these intermediates.

What is the role of NDUFAF1 in mitochondrial dysfunction associated with cancer metabolism?

NDUFAF1 has emerged as a critical link between oncogenic signaling and mitochondrial function, particularly in cancer metabolism. Researchers can employ NDUFAF1 antibodies to investigate this connection through several methodological approaches:

Proteomic analysis using stable isotope labeling with amino acids (SILAC) coupled with LC-MS has revealed that oncogenic K-Ras induction leads to significant decrease (approximately 50%) in NDUFAF1 expression . This decrease was validated in primary human pancreatic cancer tissues, establishing NDUFAF1 as a mediator of K-Ras-induced mitochondrial dysfunction.

Functional studies have demonstrated that knockdown of NDUFAF1 causes mitochondrial respiration deficiency, accumulation of NADH, and subsequent increase in glycolytic activity . This metabolic shift mirrors the Warburg effect observed in many cancers, suggesting NDUFAF1 as a potential mechanistic link between oncogenic signaling and cancer metabolism.

In experimental designs, NDUFAF1 antibodies can be used to:

• Quantify NDUFAF1 expression changes in response to oncogenic signals

• Track alterations in complex I assembly in cancer cells

• Identify potential therapeutic strategies targeting the NDUFAF1-dependent assembly pathway

• Evaluate the correlation between NDUFAF1 levels and glycolytic phenotypes in tumor samples

Western blotting using NDUFAF1 antibodies has been employed in studies examining bladder outlet obstruction, revealing HIF-1α-mediated metabolic switching and mitochondrial remodeling , further supporting the role of NDUFAF1 in metabolic adaptation.

How can researchers optimize co-immunoprecipitation protocols with NDUFAF1 antibodies?

Optimizing co-immunoprecipitation (co-IP) with NDUFAF1 antibodies requires careful consideration of several methodological factors:

First, proper mitochondrial isolation is essential before co-IP, as NDUFAF1 is a mitochondrial protein. This enrichment step reduces cytosolic contaminants and increases the signal-to-noise ratio for detecting relevant interacting partners.

Second, detergent selection is critical for solubilizing membrane-associated complexes while preserving protein-protein interactions. Published studies have successfully used mild detergents to maintain the integrity of NDUFAF1-containing complexes during immunoprecipitation .

Third, control selection is crucial for result interpretation. Published protocols demonstrate the effectiveness of using pre-immune sera as negative controls, which should not enrich any subunits . This approach helps distinguish specific interactions from background binding.

Fourth, for temporal assembly studies, pulse-chase labeling protocols have proven effective. Research demonstrates that following pulse-chase labeling of mtDNA-encoded subunits, mitochondria can be isolated and subjected to co-immunoprecipitation using antibodies directed to endogenous NDUFAF1 . This method revealed strong temporal interactions with ND2 and weaker interactions with ND1, ND3, and ND4L that decreased over time.

Fifth, for studying assembly intermediates, sequential immunoprecipitation approaches can be considered. Given that NDUFAF1 exists in different assembly complexes (approximately 600 and 700 kDa in size) , sequential IP may help distinguish the composition of these distinct intermediates.

What methodological approaches can delineate the structural role of NDUFAF1 in complex I assembly?

Recent structural studies have provided unprecedented insights into NDUFAF1's function, offering methodological frameworks for researchers:

Cryo-electron microscopy has been successfully employed to determine the structure of NDUFAF1-containing assembly intermediates . This approach revealed that NDUFAF1 locks the central ND3 subunit in an assembly-competent conformation, and major rearrangements of central subunits are required for complex I maturation.

For laboratories without access to cryo-EM, complementary biochemical approaches can still provide valuable structural insights. Blue Native PAGE analysis of samples from NDUFAF1 deletion strains showed absence of the P-P module intermediates, confirming NDUFAF1's essential role in formation of the MCIA complex . This method can be adapted to study the effects of specific NDUFAF1 mutations or domains on assembly.

Complexome profiling, a mass spectrometry-based approach, can be used to identify proteins co-migrating with NDUFAF1-containing complexes . This technique has revealed unexpected components of NDUFAF1 assemblies, such as the cardiolipin remodeling enzyme tafazzin as an integral component of the core complex.

Site-directed mutagenesis combined with functional complementation assays provides another powerful approach. Research has demonstrated that complementation with plasmid-expressed NDUFAF1 restores complex I expression in deletion strains . This system can be adapted to study specific domains or residues of NDUFAF1 critical for its assembly function.

What are common technical challenges when using NDUFAF1 antibodies and how can they be addressed?

Researchers working with NDUFAF1 antibodies may encounter several technical challenges that can be addressed through methodological refinements:

For Western blotting applications, the discrepancy between calculated (38 kDa) and observed (32-33 kDa) molecular weights of NDUFAF1 may cause confusion in band identification. This can be addressed by including positive controls such as HEK-293, Jurkat, or HeLa cell lysates, which have been validated to express detectable levels of NDUFAF1 .

In immunohistochemistry applications, optimization of antigen retrieval is critical. Protocols suggest using TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative . Each new tissue type may require optimization of these conditions.

For co-immunoprecipitation studies tracking complex I assembly, the transient nature of NDUFAF1 interactions with mtDNA-encoded subunits necessitates careful timing. Research shows strong interactions immediately after pulse labeling, decreasing at 3 hours, and virtually absent after 24 hours . Experimental designs should account for this temporal dynamics.

In studies comparing NDUFAF1 levels across different conditions, proper normalization is essential. For mitochondrial proteins like NDUFAF1, normalization to mitochondrial content markers rather than total cellular protein provides more accurate comparisons.

When analyzing complex assembly intermediates by BN-PAGE, the presence of multiple NDUFAF1-containing complexes (600 and 700 kDa) can complicate interpretation. Using multiple antibodies against different complex I components can help define the composition of these intermediates.

How can researchers integrate NDUFAF1 antibody data with functional assessments of mitochondrial activity?

Integrating structural data from NDUFAF1 antibody studies with functional mitochondrial assessments provides a comprehensive understanding of complex I biology:

First, combine Western blot quantification of NDUFAF1 and complex I subunits with enzyme activity assays. Studies have demonstrated that NDUFAF1 knockdown results in reduced NADH:ubiquinone oxidoreductase activity . This correlation helps establish the functional consequences of altered NDUFAF1 expression or complex I assembly.

Second, parallel assessment of complex I-dependent respiration using oxygen consumption measurements complements antibody-based detection of assembly states. Research has shown that NDUFAF1 deletion results in decreased NADH:hexaammineruthenium activity and undetectable ubiquinone reductase activity .

Third, for metabolic studies, measuring NADH/NAD+ ratios and glycolytic parameters alongside NDUFAF1 levels provides mechanistic insights. Knockdown studies have demonstrated that NDUFAF1 reduction leads to NADH accumulation and increased glycolytic activity , establishing a direct link between complex I assembly and metabolic reprogramming.

Fourth, in genetic studies, correlate antibody-detected NDUFAF1 levels with mutation analysis. The distribution of NDUFAF1-containing assembly intermediates has been shown to be altered in complex I deficient patients , providing potential diagnostic applications.

Fifth, for cancer metabolism studies, combine NDUFAF1 detection with oncogenic signaling pathway analysis. Research has established NDUFAF1 as a mediator of K-Ras-induced mitochondrial dysfunction , highlighting the importance of integrating mitochondrial assembly data with oncogenic signaling pathways.

How might NDUFAF1 antibodies contribute to understanding the relationship between complex I assembly and cardiolipin metabolism?

Recent structural studies have revealed an unexpected link between complex I assembly and cardiolipin metabolism that researchers can explore using NDUFAF1 antibodies:

Cryo-EM structures of NDUFAF1-containing assembly intermediates revealed that the cardiolipin remodeling enzyme tafazzin is an integral component of the core complex . This surprising finding suggests direct coordination between complex I assembly and cardiolipin metabolism, which is critical for maintaining mitochondrial membrane architecture and function.

Researchers can exploit this discovery by using NDUFAF1 antibodies to co-immunoprecipitate tafazzin and other components of this functional module. Quantitative analysis of these interactions under different physiological or pathological conditions might reveal regulatory mechanisms coordinating respiratory complex assembly with membrane phospholipid composition.

Blue Native PAGE coupled with Western blotting for both NDUFAF1 and tafazzin could track the co-migration of these proteins in assembly intermediates. Alterations in this pattern in models of mitochondrial dysfunction could provide insights into the sequence of assembly events and their dependence on proper cardiolipin metabolism.

Proximity labeling approaches using NDUFAF1 as bait, followed by validation with specific antibodies, might identify additional components of this functional module and expand our understanding of the complex I assembly mechanism and its coordination with membrane biogenesis.

What methodological approaches can assess the dynamic regulation of NDUFAF1 in response to mitochondrial stress?

Understanding how NDUFAF1 responds to mitochondrial stress conditions provides insights into adaptive mechanisms regulating complex I assembly:

Time-course analysis using NDUFAF1 antibodies following induction of mitochondrial stress (e.g., electron transport chain inhibitors, oxidative stress, hypoxia) can reveal dynamic changes in NDUFAF1 expression and localization. Western blotting, immunofluorescence, and quantitative PCR can be employed in parallel to distinguish transcriptional, translational, and post-translational regulation.

Subcellular fractionation combined with NDUFAF1 immunodetection can track potential redistribution between different mitochondrial compartments or assembly intermediates under stress conditions. This approach helps understand how complex I assembly is regulated in response to changing cellular demands or damage.

Analysis of post-translational modifications of NDUFAF1 under stress conditions may provide insights into regulatory mechanisms. Immunoprecipitation with NDUFAF1 antibodies followed by mass spectrometry can identify potential phosphorylation, acetylation, or other modifications that might regulate its function.

For metabolic adaptations, combining NDUFAF1 detection with metabolic flux analysis under different stress conditions can establish connections between complex I assembly status and cellular bioenergetic adaptations. Research has already linked NDUFAF1 reduction to increased glycolytic activity , suggesting important roles in metabolic reprogramming during stress.