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

Play Video

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).

View Video