Blue Native Polyacrylamide Gel Electrophoresis: A Non-Denaturing Separation Technique for Analysis of Intact Mitochondrial Respiratory Chain Complexes

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.

1. 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.
2. Prepare 10 mL of 2 M aminocaproic acid in dH₂O.
3. Prepare 1 mL of the mitochondrial buffer by combining the following: 0.5 mL of 3x gel buffer, 0.5 mL....

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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