Non‐Gradient Blue Native Polyacrylamide Gel Electrophoresis

Xiaoting Luo1, Jinzi Wu2, Zhen Jin2, Liang‐Jun Yan3

1 Gannan Medical University, Ganzhou, Jiangxi Province, 2 University of North Texas Health Science Center, Fort Worth, Texas, 3 Corresponding author (liang‐
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.29
DOI:  10.1002/cpps.21
Online Posting Date:  February, 2017
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Gradient blue native polyacrylamide gel electrophoresis (BN‐PAGE) is a well established and widely used technique for activity analysis of high‐molecular‐weight proteins, protein complexes, and protein‐protein interactions. Since its inception in the early 1990s, a variety of minor modifications have been made to this gradient gel analytical method. Here we provide a major modification of the method, which we call non‐gradient BN‐PAGE. The procedure, similar to that of non‐gradient SDS‐PAGE, is simple because there is no expensive gradient maker involved. The non‐gradient BN‐PAGE protocols presented herein provide guidelines on the analysis of mitochondrial protein complexes, in particular, dihydrolipoamide dehydrogenase (DLDH) and those in the electron transport chain. Protocols for the analysis of blood esterases or mitochondrial esterases are also presented. The non‐gradient BN‐PAGE method may be tailored for analysis of specific proteins according to their molecular weight regardless of whether the target proteins are hydrophobic or hydrophilic. © 2017 by John Wiley & Sons, Inc.

Keywords: blue native gel; esterase; electrophoresis; mitochondria; non‐gradient

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Table of Contents

  • Introduction
  • Basic Protocol 1: Non‐Gradient Blue Native Polyacrylamide Gel Electrophoresis for Resolving Mitochondrial Proteins
  • Support Protocol 1: Procedures for Activity Staining of Mitochondrial Electron Transport Chain Complexes and Dihydrolipoamide Dehydrogenase (DLDH)
  • Support Protocol 2: Procedures for Esterase Activity Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Non‐Gradient Blue Native Polyacrylamide Gel Electrophoresis for Resolving Mitochondrial Proteins

  • Reagents for gel preparation (Table 19.29.1)
    •  3× gel buffer (see recipe)
    •  50% acylamide/bisacrylamide solution (see recipe)
    •  10% (w/v) ammonium persulfate
    •  TEMED
  • Mitochondrial pellet (for isolation of mitochondria from rat or mouse brains, refer to Yan et al., )
  • Mitochondria extraction buffer (see recipe)
  • 10× sample buffer (see recipe)
  • Cathode buffer A (see recipe)
  • Cathode buffer B (see recipe)
  • Sonicator (Sonic Dismembrator; Fisher, cat. no. FB50110; 50 W, 110 V)
  • Additional reagents and equipment for polyacrylamide gel electrophoresis (unit 10.1; Gallagher, ), Bradford protein assay (unit 3.4; Olson and Markwell, ), and staining of gels (unit 10.5; Echan and Speicher, )
Table 9.9.1   MaterialsRecipes for Acrylamide Gels a

Resolving Resolving Stacking
8.0% (for SMP) 12% (for matrix) 4%
H 2O 5.0 ml 4.27 ml 2.9 ml
Gel buffer (3×) 3.33 ml 3.33 ml 1.67 ml
Acrylamide (50%) 1.6 ml 2.4 ml 0.4 ml
APS (10%) 50 μl 50 μl 25 μl
TEMED 7 μl 7 μl 7.5 μl
Total volume 10 ml 10 ml 5 ml

 aFor gel percentages other than the two given in the table, one can vary the volume of the 50% acrylamide solution and the volume of water. The concentrations of the remaining components remain the same. For example, if a 9% separating gel is to be made, the volume of the 50% acrylamide would be 1.8 (= 9 × 10/50) when the total volume is 10 ml, and the volume of water would be 4.8 ml. The percentage of the stacking gel is always 4%.

Support Protocol 1: Procedures for Activity Staining of Mitochondrial Electron Transport Chain Complexes and Dihydrolipoamide Dehydrogenase (DLDH)

  • Gel strip (cut from the gel prepared in protocol 1Basic Protocol 1) containing mitochondrial electron transport chain complexes or dihydrolipoamide dehydrogenase (DLDH): the gel strip should not be stained with Coomassie blue or destained, as these steps will denature the enzymes on the gel strip; use the strip as it is immediately after gel electrophoresis
  • Nitroblue tetrazolium tablets (10 mg/tablet from Sigma)
  • NADH (Sigma)
  • 50 mM potassium phosphate buffer, pH 7.0 ( appendix 2E)
  • 5 mM Tris·Cl, pH 7.4 ( appendix 2E)
  • Sodium succinate
  • Phenazine methosulfate (Sigma, cat. no. P9625; stock solution prepared in DMSO)
  • 50 mM sodium phosphate buffer, pH 7.2 ( appendix 2E)
  • 3, 3′‐diaminobenzidine tetrachloride (DAB)
  • Cytochrome c
  • Tris‐glycine buffer (35 mM Tris, 270 mM glycine, pH 8.3)
  • MgCl 2
  • Pb(NO 3) 2
  • ATP

Support Protocol 2: Procedures for Esterase Activity Staining

  • Gel strips: prepared following gel electrophoresis ( protocol 1Basic Protocol) by cutting each lane out of the slab gel
  • 50 mM Tris·Cl, pH 7.4 ( appendix 2E)
  • α‐ or β‐naphthyl acetate (Sigma, cat. no. N8505 or N6875, respectively)
  • Ethanol
  • Fast blue BB (Sigma‐Aldrich, cat. no. 44670)
  • Acetic acid
  • Methanol
NOTE: This esterase activity staining method can be used to analyze esterase activities from a variety of sources such as serum, mitochondria, and cytosol. Regardless of the source of the enzymes to be analyzed, the final protein concentration in the loading sample should usually be 1 to 2 mg/ml. Gel running conditions are the same as described in the protocol 1Basic Protocol for mitochondrial samples.
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Literature Cited

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