Blue Native Polyacrylamide Gel Electrophoresis: A Non-Denaturing Separation Technique for Analysis of Intact Mitochondrial Respiratory Chain Complexes
Published: April 30, 2023
Abstract
Source: Konovalova, S. Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting. J. Vis. Exp. (2019)
In this video, we demonstrate an electrophoretic technique, called blue native PAGE, for the analysis of mitochondrial respiratory protein complexes in their natively folded and functionally active state. The anionic Coomassie blue dye causes the protein complexes to become negatively charged, allowing separation based on size and structure.
Protocol
1. Preparation of buffers and solutions NOTE: All the buffers and solutions used in the protocol are summarized in Table 1. The given volumes of the buffers are sufficient to prepare and run 10 samples. All the buffers can be stored at +4 °C for up to one year. Prepare 20 mL of 3x gel buffer containing 1.5 M aminocaproic acid, 150 mM Bis-tris in distilled water (dH2O). Adjust pH to 7.0. Prepare 10 mL of 2 …
Representative Results
Table 1: Buffers and solutions used for BN-PAGE Buffer or solution Composition Recipe 3 x gel buffer 1.5 M aminocaproic acid 3.94 g of aminocaproic acid 150 mM Bis-tris 0.63 g of Bis-tris <td…
Disclosures
The authors have nothing to disclose.
Materials
| 100 mm cell plates | Thermo Fisher Scientific | 130182 | |
| 40% Acrylamide/Bis 37.5:1 | Bio-Rad | 161-0148 | |
| Aminocaproic acid | Sigma | A2504 | |
| Ammonium persulfate | Sigma | A3678 | |
| Bis-tris | Sigma | B7535 | |
| Bradford protein assay kit | BioRad | 5000006 | |
| Cell scrapers | Fisher | 11597692 | |
| Coomassie blue G250 (Serva Blue G) | Serva | 35050 | |
| Digitonin | Sigma | D141 | |
| DMEM | Lonza | 12-614F/12 | |
| EDTA | Life Technologies | 15575-038 | |
| FBS | Life Technologies | 10270106 | |
| Fetal bovine serum | Life Technologies | 10270106 | |
| Glycerol | Sigma | G5516 | |
| Gradient maker | Bio-Rad | 1654120 | |
| Lauryl maltoside (n-Dodecyl β-D-maltoside) | Sigma | D4641 | |
| L-glutamine | Life Technologies | 25030024 | |
| L-glutamine | Gibco | 25030 | |
| N,N,N′,N′-Tetramethylethylenediamine (TEMED) | Sigma | T9281 | |
| PBS | Life Technologies | BE17-516F | |
| Penicillin/Streptomycin | Lonza | 17-602E | |
| Penicillin/Streptomycin | Gibco | 15140 | |
| Power supply | GE Healthcare | EPS 301 | |
| Protease inhibitors | Thermo Scientific | 10137963 | |
| Trans-blot turbo mini PVDF | BioRad | 1704156 | |
| Tricine | Sigma | T9784 | |
| Trypsin-EDTA | Gibco | 15400054 | |
| Vertical electrophoresis apparatus | BioRad | 1658004 |
Cite This Article
Blue Native Polyacrylamide Gel Electrophoresis: A Non-Denaturing Separation Technique for Analysis of Intact Mitochondrial Respiratory Chain Complexes. J. Vis. Exp. (Pending Publication), e21110, doi: (2023).