blue native PAGE is very interesting

Use Coomassie Blue G (Sigma B0770) to stain the protein sample first, then run the native gel
After the electrophoresis, the protein complex is still active! You can transfer it for immunoblotting, or do some kind in-gel enzyme activity assay. Very nice.

Pediatr Res. 2001 Nov;50(5):658-65.
Blue native polyacrylamide gel electrophoresis: a powerful tool in diagnosis of oxidative phosphorylation defects.
Department of Pediatrics, Division of Pediatric Neurology and Metabolism, Ghent University Hospital, De Pintelaan, 185, 9000 Gent, Belgium. rudy.vancoster@rug.ac.be

Catalytic activity of oxidative phosphorylation complexes is maintained following separation by Blue Native polyacrylamide gel electrophoresis (BN-PAGE). In BN-PAGE gels, using histochemical staining methods, we have demonstrated enzymatic activity of the complexes I, II, IV, and V in heart and skeletal muscle, liver, and cultured skin fibroblasts. The combination of BN-PAGE and catalytic staining can be successfully applied for detection of complex deficiencies. Tissues from 18 patients with deficiency in the oxidative phosphorylation as detected by spectrophotometric assay were used (10 patients complex IV, three patients complex I, one patient complex II, one patient complex I+III, three patients complex I+IV). The gene defect was located in nuclear DNA in five patients and mitochondrial DNA in one patient. In samples from patients with a severe deficiency, almost complete absence of the corresponding enzyme band is observed after catalytic staining in the gel. In patients with known partial deficiency, a milder decrease of the corresponding enzyme band is demonstrated. The amount of protein in complexes I, V, and III can easily be evaluated in samples from heart and skeletal muscle after separation by BN-PAGE using silver or Coomassie staining. The protein amount in complex IV is difficult to visualize by silver staining but easier by the Coomassie technique. In samples from liver and cultured skin fibroblasts, evaluation of protein amount is more difficult due to high background staining. In these tissues, immunoblotting can be done after BN-PAGE and subsequent transfer to a nitrocellulose membrane.

http://www.mitosciences.com/ms603.html

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 a 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.