Review ArticleElectrophoresis techniques to investigate defects in oxidative phosphorylation
Introduction
Defects in the oxidative phosphorylation (OXPHOS) system give rise to numerous neuromuscular disorders [[1], Bonilla and Tanji, this issue]. The huge clinical differences in phenotype, which can vary from deafness to early infant death, make these mitochondrial disorders difficult to identify and characterize. Nonetheless, with an incidence of at least 1 in 5000 individuals it is recognized as the most common group of inborn errors of metabolism [2]. When a patient is clinically diagnosed, specialized enzyme assays are performed to find possible deficiencies of the OXPHOS complexes. This can results in a defect of a single OXPHOS complex (called isolated complex deficiency) or in a defect of more enzymes at the same time (multiple complex deficiency). The next challenge is to identify the disease gene, which is very complicated because over two hundred genes from both mitochondrial and nuclear DNA can be involved [Aigar et al., this issue]. Even when just a single enzyme is affected numerous genes remain to be sequenced. In this article we describe how electrophoresis techniques can aid the complicated task of finding the disease-causing mutation in these patients.
The OXPHOS system is the main ATP generating system in our cells. It uses the energy released by the reduction of substrates to build up a proton gradient across the mitochondrial inner membrane, which on its turn is used by complex V to convert ADP into the high energy compound ATP [Marcinek et al., this issue]. The five multi-subunit complexes consist of many subunits, which are encoded by either mitochondrial or nuclear DNA. Electrophoretic separation of these complexes is a complicated task for several reasons. Firstly, due to the fact that the OXPHOS complexes are integral membrane proteins, they are very hydrophobic and therefore require the addition of detergents to keep them in solution. Secondly, because they are multi-subunit complexes there is risk of partial dissociation during the electrophoresis process. Thirdly, it is believed that the complexes are not single entities but are organized in higher order structures called supercomplexes [3]. Therefore the concentration and type of detergent used are of crucial importance for the outcome of native electrophoresis experiments. Schagger and Von Jagow have found a good way to deal with these problems by introducing Blue native electrophoresis (BN-PAGE) [4]. This technique combines the mild properties of the detergent n-dodecyl β-d-maltoside (DDM) to dissolve the native protein complexes in combination with the use of the anionic dye Coomassie brilliant blue G250 to introduce a charge shift required for the mobility of the protein complexes in the electric field. The sieving achieved by the correct gradient of a polyacrylamide matrix results in a optimal separation of the mitochondrial OXPHOS complexes in their native form.
After its introduction, this method has been widely used and many variations and adaptations for specific research questions have been implemented. For instance, the use of the mild detergent digitonin instead of DDM has led to the identification and separation of OXPHOS supercomplexes [3]. Also Clear native electrophoresis (CN-PAGE) has been introduced for improvement of downstream applications such as mass spectrometry, in-gel activity staining or fluorescence resonance energy transfer [5]. Instead of being encyclopedic on all of the “ins and outs” of native electrophoresis, in this article we present the basic technique with a focus on elucidating defects in patients with OXPHOS deficiencies. More detailed reading on this subject is available [6], [7], [8]).
From patients with a suspected mitochondrial OXPHOS disorder, routinely fibroblast or muscle biopsies are obtained to perform photo-spectrometric enzyme activity assays or metabolic assays. However, small amounts of these tissues can also be used for BN-PAGE analysis, which is good pre-screening for possible defects in the OXPHOS system. This easily applicable technique can be performed using standard electrophoresis devices available in most laboratories. After a first dimension blue native gel in combination with in-gel activity assays and western blotting using antibodies against the OXPHOS complexes many defects can be readily observed. To obtain further information on the assembly status of the affected complex, two dimensional Blue native/SDS–PAGE can be performed. In many instances, this will also yield additional information about the nature of the deficiency and the specific subunit affected.
Section snippets
Samples preparation
The most commonly available patient materials are muscle biopsies or in vitro cultured skin fibroblasts. Therefore we describe the basic protocol using these as a starting materials. The protocol we use for fibroblasts [9] or muscle biopsies [10] is a trade-off between the purity of the sample and the amount of tissue. Enriched mitochondrial fractions are of sufficient purity to obtain good BN-PAGE results and have the advantage that limited amounts of precious patient tissue can be used.
Blue native polyacrylamide gel electrophoresis
We normally use a minigel system to perform the electrophoresis (Bio-Rad Mini-Protean 3). This size is sufficient for a good separation of the OXPHOS complexes and has the advantage that it requires less reagents and less patient material. This becomes even more important considering down stream applications such as Western blotting and in-gel activity assays, which necessitate expensive chemicals and antibodies. The casting of a gradient gel is the most delicate part of this procedure. For
Two dimensional blue native/SDS electrophoresis
As the first dimension blue native PAGE separates intact OXPHOS complexes, subsequent denaturing electrophoresis resolves the individual subunits of the respective complexes. This way the content and distribution of the individual subunits can be displayed, allowing the detection of possible subassemblies. Such subassemblies can in some cases be indicative for the presence of a specific gene defect (see Section 5). Another advantage of a second dimension gel in combination with western blotting
Interpretation of results
The cartoon in Fig. 2C shows the principle of a 2D BN/SDS gel. When performing a Western blot using a complex-subunit specific antibody, the most intense spot occurs at the place where in the first dimension the complex has ran. However, such a subunit can also appear at lower molecular weights (from the first dimension). This way several spots from the same antibody occur on the same horizontal line (Fig. 2C). These lower molecular weight signals indicate that not all of this subunit is
Concluding remarks
Almost 17 years after its inception, BN-PAGE been a widely used technique in the mitochondrial field as illustrated by the fact that the first article by Schagger and Von Jagow has been cited to date over 725 times [4]. Since then Schagger and others have continued to refine the method and explored new applications. Several excellent reviews about this method have been published, which are recommended for background information [6], [26], [8], [27], [28].
This article is a hands-on description
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2020, STAR ProtocolsCitation Excerpt :As the first dimension BN-PAGE separates intact OXPHOS complexes, subsequent denaturing electrophoresis in the presence of the anionic detergent sodium dodecyl sulfate (SDS) resolves the individual subunits of the respective complexes and supercomplexes. In combination with immunoblotting, it offers the advantage that the signals are usually much stronger and that all proteins can be detected due to increased epitope availability (Calvaruso et al., 2008; Nijtmans et al., 2002). To perform a 2D-BN/SDS-PAGE analysis, following the first BN-PAGE dimension, a lane corresponding to a cell line or mitochondrial sample, is cut out of the gel with a razorblade and placed on a small plastic container for further processing.
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2018, Neuromuscular DisordersCitation Excerpt :Results were expressed as the difference between the basal fluorescence values and the increase in rhodamine 123 fluorescence levels following addition of oligomycin plus FCCP. Samples were processed according to the protocol described elsewhere [47]. Briefly, following supplementation of samples with BN-sample buffer, the molecular weight marker (NativeMARK Unstained Protein Standard, Life Technologies) and 30 µg of samples were loaded into polyacrylamide gels and run at 80V at 4 °C.