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Brief Description & Uses Blue Native polyacrylamide gel electrophoresis, BN-PAGE (1), is a simple and effective way to subfractionate mitochondrial proteins as intact complexes on a single gel (in one dimension). It can be used to detect altered assembly of these complexes arising from mutations in subunits, mutations in assembly factors, or mtDNA depletion. This type of analysis has been performed with biopsy samples, platelets and fibroblast cells from patients with suspected mitochondrial diseases. For a detailed protocol for BN-PAGE click here. In this method multisubunit enzymes bind a charged dye Coomassie brilliant blue which allows their electrophoretic separation in the first dimension by the size of the complex. Complexes I-V with masses ranging from 950K to 200K are well resolved in the first dimension. The separated proteins can then be transferred to nitrocellulose membrane/PVDF by electrophoresis and Complexes I, II, III, IV and V can be detected by mAbs against CI-NDUFA9 (MS111), CII-70 kDa subunit (MS204), CIII-Core protein 2 (MS304), CIV-subunit IV (MS407) and CV-α subunit (MS507) respectively. Such one dimensional gels, are best analyzed by using single mAbs against each complex. Other Complex I mAbs are available for BNPAGE, specifically anti-GRIM-19 (MS103) and anti-20 kDa (MS105). A sample of purified bovine heart mitochondria can be supplied with these antibodies to act as a BNPAGE control sample upon request. Sometimes a greater separation of enzymes is necessary - it is possible to separate the proteins within each individual complex. To do this, blotting is NOT performed after the first (NATIVE) dimension, instead gels are turned 90 degrees and run in a perpendicular second dimension which is denaturing (NON-NATIVE). In this way the protein subunits within each complex are separated. MitoSciences provides an optimized pre-mixed cocktail of the mAbs to SIMULTANEOUSLY detect Complexes I-V after 2nd dimension blotting (specifically the cocktail contains MS111, MS204, MS304, MS407 and MS507). An example of this kind of analysis in fibroblasts is shown in the figure below, also see examples of the use of BN-PAGE in patient case studies (link below). For a detailed description of 1st and 2nd dimension BN-PAGE click here. This cocktail is supplied with a sample of bovine heart mitochondria to act as a BNPAGE control sample. Click here to review case studies for this product. Specifications
Kit Components
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Have you published research using this product? Let us know and earn a $100.00 discount on your next purchase. References for Oxidative Phosphorylation
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