Detection and Analysis of Protein‐Protein Interactions of Organellar and Prokaryotic Proteomes by Blue Native and Colorless Native Gel Electrophoresis

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Native gels enable the analysis of protein complexes on a proteome?wide scale in a single experiment. The protocols described in this unit are based on separation of protein complexes by blue native polyacrylamide electrophoresis (BN?PAGE), the most versatile native gel system, and the closely related milder colorless native PAGE (CN?PAGE). Both BN?PAGE and CN?PAGE are described on analytical to preparative scales. In addition, methods for subsequent analysis of protein complexes are given, including electroelution from native gels as well as denaturing and native two?dimensional PAGE. Finally, the removal of Coomassie dye from electroeluted proteins is detailed along with a discussion of fundamental considerations for the solubilization of membrane protein complexes from various biological samples, which are exemplified for mitochondria, chloroplasts (thylakoids), and cyanobacteria. Curr. Protoc. Protein Sci. 54:19.18.1?19.18.36. © 2008 by John Wiley & Sons, Inc.

Keywords: protein?protein interaction; membrane proteins; detergents; solubilization; mitochondria; bacteria; electroelution

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Introduction
Strategic Planning
Basic Protocol 1: Standard Native Gels for High‐Resolution Separation of Protein Complexes
Alternate Protocol 1: Mini‐Native Gels for Fast Separation of Protein Complexes
Alternate Protocol 2: Preparative Native Gels for Isolation of Up to Milligram Amounts of Protein Complexes
Basic Protocol 2: Electroelution for Recovery of Proteins from Native Polyacrylamide Gels
Basic Protocol 3: Two‐Dimensional Blue‐Native Polyacrylamide Gel Electrophoresis (BN‐PAGE) to Separate Constituting Subcomplexes of Protein Complexes Retained in First‐Dimension Native Gels
Basic Protocol 4: Tricine‐SDS‐PAGE for Separation of Protein Complex Subunits in Second or Third Dimension
Support Protocol 1: Removal of Coomassie Dye from Electroeluted Proteins
Support Protocol 2: Solubilization of Biological Membranes for Native Gel Electrophoresis
Support Protocol 3: Construction of the H‐Shaped Elution Device and the Electroelution Chamber
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Standard Native Gels for High‐Resolution Separation of Protein Complexes

Materials

Separating and sample gel solutions (Table 19.18.1 )
Samples of soluble proteins or solubilized membrane proteins (see protocol 8 and )
Native standard proteins (e.g., in the range of 100‐ to 1000‐kDa)
Sample buffer (see reciperecipes )
Anode and cathode buffers (see reciperecipes )

Electrophoresis apparatus: Protean II 16‐cm cell (Bio‐Rad) or SE 600 16‐cm unit (Hoefer) with clamps, glass plates, casting unit, and buffer chambers
1.5‐mm spacers
Peristaltic pump and 0.110‐in. i.d., 2.79‐mm PVC tubing
19‐G needles (8‐in. long; 1.00 × 200–mm) with Luer‐connection (e.g., Rometsch GmbH or Popper & Sons)
Gradient maker with two chambers of 70‐ to 250‐ml volume (e.g., CBS Scientific GM‐150 or GM‐200 or Hoefer SG500)
Magnetic stirrers and stir bars
Graduated cylinder
Adhesive tape
1.5‐mm Teflon combs (10‐, 15‐, or 20‐tooth comb)
Constant current power supply (500 V and higher)

Clear, robust plastic foil (e.g., pieces of freezer bags or clear disposal bags—polyethylene or polypropylene, 40‐ to 50‐µm thick)

Table 9.8.1 Materials Recipes for Standard Size Polyacrylamide Separating and Stacking Gels a Recipes for Standard Size Polyacrylamide Separating and Stacking Gels

Light Acrylamide Gel Solutions for Standard Size Gradient Gels
Acrylamide concentration of light gel solution (%) b
Stock solution c	3	3.5	4	4.5	5	6
Water d	8.79 ml	8.6 ml	8.42 ml	8.23 ml	8.05 ml	7.67 ml
Light gel buffer	5 ml	5 ml	5 ml	5 ml	5 ml	5 ml
Gel A/B	1.02 ml	1.18 ml	1.35 ml	1.52 ml	1.69 ml	2.03 ml
Gel A	110 µl	129 µl	147 µl	165 µl	183 µl	221 µl
TEMED	7.64 µl	7.5 µl	7.36 µl	7.23 µl	7.09 µl	6.82 µl
10% APS	76.4 µl	75 µl	73.6 µl	72.3 µl	70.9 µl	68.2 µl
Heavy Acrylamide Gel Solutions for Standard Size Gradient Gels
	Acrylamide concentration of heavy gel solution (%) b
Stock solution c	11	13	14	16	18	20
Water d	5.81 ml	5.07 ml	4.7 ml	3.95 ml	3.21 ml	2.47 ml
Heavy gel buffer	5 ml	5 ml	5 ml	5 ml	5 ml	5 ml
Gel A/B	3.72 ml	4.40 ml	4.74 ml	5.41 ml	6.09 ml	6.77 ml
Gel A	404 µl	478 µl	514 µl	587 µl	662 µl	735 µl
TEMED	5.45 µl	4.91 µl	4.64 µl	4.09 µl	3.55 µl	3 µl
10% APS	54.5 µl	49.1 µl	46.4 µl	40.9 µl	35.5 µl	30 µl
Sample Gel Solutions
Acrylamide concentration of sample gel solution (%) b
Stock solution c	3	3.5	4	4.5	5
Water d	5.83 ml	5.70 ml	5.58 ml	5.46 ml	5.33 ml
Light gel buffer	3.33 ml	3.33 ml	3.33 ml	3.33 ml	3.33 ml
Gel A/B	677 µl	789 µl	902 µl	1.02 µl	1.13 µl
Gel A	73 µl	86 µl	98 µl	110 µl	122 µl
TEMED	8.15 µl	8 µl	7.85 µl	7.71 µl	7.56 µl
10% APS	81.5 µl	80 µl	78.5 µl	77.1 µl	75.6 µl
Reagents used in Gels
Gel A
40% (w/v) acrylamide solution (e.g., Sigma cat. no. A4058, Bio‐Rad cat. no. 161‐0141, or VWR 1.00633.1000). Store up to 2 months at 4°C.
Gel A/B
40% (w/v) acrylamide/N,N′ ‐methylene bis‐acrylamide solution 29:1 (e.g., Sigma cat. no. A7802, Serva cat. no. SERA10680.03, or Carl Roth cat. no. A515.1). Store up to 2 months at 4°C. CAUTION: It is recommended to employ ready‐to‐use solutions rather than working with the neurotoxic acrylamide powder. Always wear gloves while handling these solutions in an extractor hood and use a pipetting aid.
TEMED
10% (w/v) ammonium persulfate (APS)
Prepare a 50‐ml stock solution, dispense into 1‐ml aliquots, and store up to 2 months at −20°C. Always use freshly thawed solution.
Light gel buffer BT
7.85 g Bis‐Tris (0.15 M final) 19.68 g 6‐aminohexanoic acid (0.6 M final) Dissolve in 180 ml water. Adjust to pH 7.0 with 5 to 6 N HCl. Add water to 250 ml total volume. Filter the solution through a 0.45‐µm filter and store up to 1 month at 4°C.
Light gel buffer imi
1.27 g imidazole (75 mM final) 49.19 g 6‐aminohexanoic acid (1.5 M final) Dissolve in 180 ml water. Adjust to pH 7.0 with 5 to 6 N HCl. Add water to 250 ml total volume. Filter the solution through a 0.45‐µm filter and store up to 1 month at 4°C.
Heavy gel buffer BT
7.85 g Bis‐Tris (0.15 M final) 19.68 g 6‐aminohexanoic acid (0.6 M final) 150 g glycerol (60% w/v final) Dissolve in 60 ml water. Adjust to pH 7.0 with 5 to 6 N HCl. Add water to 250 ml total volume. Filter the solution through a 0.45‐µm filter and store up to 1 month at 4°C.
Heavy gel buffer imi
1.27 g imidazole (75 mM final) 49.19 g 6‐aminohexanoic acid (1.5 M final) 150 g glycerol (60% (w/v) final) Dissolve in 50 ml water. Adjust to pH 7.0 with 5 to 6 N HCl. Add water to 250 ml total volume. Filter the solution through a 0.45‐µm filter and store up to 1 month at 4°C.

^a The recipes produce 15 ml of each separating gel solution and 10 ml of sample gel, which are sufficient for a single gel with 1.5‐mm × 14‐cm × 16‐cm dimensions (Hoefer). When using Bio‐Rad equipment (1.5‐mm × 16‐cm × 16‐cm) or other, the respective volumes have to be adapted adequately.

^b The content of the cross‐linker N,N′ ‐methylene bis‐acrylamide is 3% in each gel.

^c Use Milli‐Q‐purified water or equivalent in all recipes and protocol steps.

^d For CN‐PAGE, various detergents can be optionally included in the gels by adding corresponding volumes of 10% detergent stock solutions that reduces accordingly the volume of water (DDM and Triton X‐100 at a final concentration of 0.01%, digitonin at a final concentration of 0.003% to 0.02%) to minimize potential aggregation of membrane proteins.

Alternate Protocol 1: Mini‐Native Gels for Fast Separation of Protein Complexes

Separating and sample gel solutions (Table 19.18.2 )
Electrophoresis apparatus for mini gels: Mini‐Protean Tetra‐cell (Bio‐Rad) or SE 260 unit (Hoefer) with clamps, glass plates, casting unit, and buffer chambers
Peristaltic pump and 0.073‐in. i.d., 1.85‐mm PVC tubing
Gradient maker with two chambers of 10‐ and 25‐ml volume
19‐G needle (4 ¾ ‐in. long; 1.00 × 120–mm) with Luer‐connection
Constant current power supply (300 V and higher)

Resealable plastic bags

Table 9.8.2 Additional Materials (also see protocol 1 ) Additional Materials Recipes for Polyacrylamide Separating and Stacking Gels e Recipes for Polyacrylamide Separating and Stacking Gels

Light Acrylamide Gel Solutions for Gradient Mini Gels
	Acrylamide concentration of light gel solution (%) f
Stock solution g	3	3.5	4	4.5	5	6
H₂ O h	4.11 ml	4.02 ml	3.93 ml	3.84 ml	3.76 ml	3.59 ml
Light gel buffer	2.33 ml	2.33 ml	2.33 ml	2.33 ml	2.33 ml	2.33 ml
Gel A/B	474 µl	553 µl	632 µl	711 µl	789 µl	947 µl
Gel A	51 µl	60 µl	68 µl	77 µl	86 µl	103 µl
TEMED	3.56 µl	3.5 µl	3.44 µl	3.37 µl	3.31 µl	3.18 µl
10% APS	35.6 µl	35 µl	34.4 µl	33.7 µl	33.1 µl	31.8 µl

Heavy Acrylamide Gel Solutions for Gradient Mini Gels
	Acrylamide concentration of heavy gel solution (%) f
Stock solution g	11	13	14	16	18	20
H₂ O h	2.72 ml	2.37 ml	2.20 ml	1.85 ml	1.50 ml	1.15 ml
Heavy gel buffer	2.33 ml	2.33 ml	2.33 ml	2.33 ml	2.33 ml	2.33 ml
Gel A/B	1.74 ml	2.05 ml	2.21 ml	2.53 ml	2.84 ml	3.16 ml
Gel A	188 µl	223 µl	240 µl	275 µl	309 µl	343 µl
TEMED	2.55 µl	2.29 µl	2.16 µl	1.91 µl	1.65 µl	1.4 µl
10% APS	25.5 µl	22.9 µl	21.6 µl	19.1 µl	16.5 µl	14 µl

Sample Gel Solutions
	Acrylamide concentration of sample gel solution (%) f
Stock solution g	3%	3.5	4	4.5	5
H₂ O h	2.33 ml	2.28 ml	2.24 ml	2.19 ml	2.13 ml
Light gel buffer	1.33 ml	1.33 ml	1.33 ml	1.33 ml	1.33 ml
Gel A/B	271 µl	316 µl	361 µl	406 µl	451 µl
Gel A	29 µl	34 µl	39 µl	44 µl	49 µl
TEMED	3.26 µl	3.2 µl	3.14 µl	3.08 µl	3.03 µl
10% APS	32.6 µl	32 µl	31.4 µl	30.8 µl	30.3 µl
Reagents used in Gels
See protocol 1 and Table 19.18.1 .

^e The recipes produce 7 ml of each separating gel solution and 4 ml of sample gel, which are sufficient for mini gels with 1.5‐mm × 8.3‐cm × 7.3‐cm (Bio‐Rad) to 1.5‐mm × 8.2‐cm × 9.7‐cm (Hoefer) dimensions.

^f The content of the cross‐linker N,N′ ‐methylene bis‐acrylamide is 3% in each gel.

^g All reagents and solutions in the protocol must be prepared with Milli‐Q‐purified water or equivalent.

^h For CN‐PAGE, various detergents can be optionally included in the gels by adding corresponding volumes of 10% detergent stock solutions, which reduces accordingly the volume of water (DDM and Triton X‐100 at a final concentration of 0.01%, digitonin at a final concentration of 0.003% to 0.02%) to minimize potential aggregation of membrane proteins.

Alternate Protocol 2: Preparative Native Gels for Isolation of Up to Milligram Amounts of Protein Complexes

Separating and sample gel solutions (Table 19.18.3 )
Electrophoresis apparatus: Protean II 16‐cm cell (Bio‐Rad) or SE 600 ruby dual cooled vertical unit (Hoefer) with clamps, glass plates, casting unit, and buffer chambers
Thick spacers: 3‐mm spacers for the Bio‐Rad apparatus or for the Hoefer unit, conventional parts should be used as templates by a skilled workshop to produce thick spacers from 3‐, 4‐, 5‐, or 6‐mm standard PVC plates
Peristaltic pump and 0.110‐in. i.d., 2.79‐mm PVC tubing
Gradient maker with two chambers of 70‐ and 250‐ml volume
19‐G needle (8‐in. long, 1.00 × 200–mm) with Luer connection
Thick Teflon combs for one small reference well and one broad preparative well
Recirculating chiller with at least 250 W cooling capacity

Constant current power supply (500 V and higher)

Table 9.8.3 Additional Materials (also see protocol 1 ) Additional Materials Recipes for Polyacrylamide Separating and Stacking Gels j Recipes for Polyacrylamide Separating and Stacking Gels

Light Acrylamide Gel Solutions for Preparative Gradient Gels
	Acrylamide concentration of light gel solution (%) k
Stock solution l	3	3.5	4	4.5	5
H₂ O	19.93 ml	19.51 ml	19.09 ml	18.66 ml	18.24 ml
Light gel buffer	11.33 ml	11.33 ml	11.33 ml	11.33 ml	11.33 ml
Gel A/B	2.30 ml	2.68 ml	3.07 ml	3.45 ml	3.84 ml
Gel A	250 µl	292 µl	333 µl	375 µl	416 µl
TEMED	17.31 µl	17 µl	16.69 µl	16.38 µl	16.07 µl
10% APS	173.1 µl	170 µl	166.9 µl	163.8 µl	160.7 µl

Heavy Acrylamide Gel Solutions for Preparative Gradient Gels
	Acrylamide concentration of heavy gel solution (%) k
Stock solution l	7	9	11	12	13	15
H₂ O	16.56 ml	14.87 ml	13.18 ml	12.34 ml	11.5 ml	9.81 ml
Heavy gel buffer	11.33 ml	11.33 ml	11.33 ml	11.33 ml	11.33 ml	11.33 ml
Gel A/B	5.37 ml	6.90 ml	8.44 ml	9.20 ml	9.97 ml	11.50 ml
Gel A	583 µl	750 µl	916 µl	999 µl	1.08 ml	1.25 ml
TEMED	14.84 µl	13.6 µl	12.36 µl	11.75 µl	11.13 µl	9.89 µl
10% APS	148.4 µl	136 µl	123.6 µl	117.5 µl	111.3 µl	98.9 µl

Sample Gel Solutions
	Acrylamide concentration of sample gel solution (%) k
Stock solution l	3	3.5	4	4.5	5
H₂ O	11.65 ml	11.41 ml	11.16 ml	10.91 ml	10.67 ml
Light gel buffer	6.67 ml	6.67 ml	6.67 ml	6.67 ml	6.67 ml
Gel A/B	1.35 ml	1.58 ml	1.80 ml	2.03 ml	2.26 ml
Gel A	147 µl	171 µl	195 µl	221 µl	245 µl
TEMED	16.29 µl	16 µl	15.71 µl	15.42 µl	15.13 µl
10% APS	162.9 µl	160 µl	157.1 µl	154.2 µl	151.3 µl
Reagents used in Gels
See protocol 1 and Table 19.18.1 .

^J The recipes produce 34 ml of each separating gel solution and 20 ml of sample gel, which are adequate for one preparative gel with 3‐mm × 16‐cm × 16‐cm dimensions (Bio‐Rad). When using other spacers or different equipment, the respective volumes must be adapted adequately.

^k The content of the crosslinker N,N′ ‐methylene bis‐acrylamide is 3% in each gel.

^l All reagents and solutions in the protocol must be prepared with Milli‐Q‐purified water or equivalent.

Basic Protocol 2: Electroelution for Recovery of Proteins from Native Polyacrylamide Gels

Materials

1% (w/v) SDS
Electroelution buffer (see reciperecipes )

2‐kDa dialysis membrane tubing (Accurate Chemical & Scientific cat. no. 50307)
Blotting filter paper (15 × 15–cm minimum size)
Robust plate (e.g., 4‐cm thick wooden plate)
Hole punch (1.8‐ and 2.5‐cm diameter circles for each of the lower ends of both vertical tubes of the H‐shaped device)
H‐shaped elution device made according to Hunkapiller et al. ( ) or equivalent (available from C.B.S. Scientific ECU‐040‐20: electroeluter/concentrator with four 2‐ml blocks or 5‐ml blocks) (Fig. 19.18.3 ; for in‐laboratory construction details, see protocol 9 )
Elution tank for H‐shaped eluter (see protocol 9 )
5‐ to 20‐ml syringes
Razor blades
Tweezers
Power supply

Basic Protocol 3: Two‐Dimensional Blue‐Native Polyacrylamide Gel Electrophoresis (BN‐PAGE) to Separate Constituting Subcomplexes of Protein Complexes Retained in First‐Dimension Native Gels

Materials

Anode and cathode buffers (see reciperecipes )
First dimension BN‐gel containing the protein bands of sample
Separating and sample gel solutions (Table 19.18.1 )
Native standard proteins (e.g., in the range of 100 to 1000 kDa)
Sample buffer (see reciperecipes )

Clear, robust plastic foil (e.g., pieces of freezer bags or clear disposal bags—polyethylene or polypropylene, 40‐ to 50‐µm thick)
Electrophoresis apparatus: Protean II 16‐cm cell (Bio‐Rad) or SE 600 16‐cm unit (Hoefer) with clamps, glass plates, casting stand, and buffer chambers
1.5‐mm spacers
Peristaltic pump and 0.110‐in. i.d., 2.79‐mm PVC tubing
19‐G needles (8‐in. long; 1.00 × 200–mm) with Luer‐connection (e.g., Rometsch GmbH or Popper & Sons)
Gradient maker with two chambers of 70‐ to 250‐ml volume (e.g., CBS Scientific GM‐150 or GM‐200 or Hoefer SG500)
Magnetic stirrers and stir bars
Graduated cylinder
Adhesive tape
1.5‐mm Teflon combs with 10, 15, or 20 teeth
Constant current power supply (500 V and higher)

Basic Protocol 4: Tricine‐SDS‐PAGE for Separation of Protein Complex Subunits in Second or Third Dimension

Materials

Native first or second dimension gel (see Basic Protocols protocol 11 and protocol 53 and protocol 2 )
1% SDS or a 1% SDS/1% mercaptoethanol solution
Separating and stacking gel solutions (Table 19.18.4 )
Denatured standard proteins
Tricine gel anode and cathode buffer (see reciperecipes )

Scalpel or plastic ruler
Clear, robust plastic foil (e.g., pieces of freezer bags or clear disposal bags—polyethylene or polypropylene, 40‐ to 50‐µm thick)
Extractor hood
Pasteur pipets
Electrophoresis apparatus: Protean II 16‐cm cell (Bio‐Rad) or SE 600 16‐cm unit (Hoefer) with clamps, glass plates, casting stand, and buffer chambers
1.5‐mm spacers
1.5‐mm Teflon combs with 10, 15, or 20 teeth

Constant current power supply (500 V and higher)

Table 9.8.4 Materials Recipes for Standard Size Tricine Polyacrylamide Separating and Stacking Gels m Recipes for Standard Size Tricine Polyacrylamide Separating and Stacking Gels

	Acrylamide concentration of separating gel (%) n
Stock solution	10	13	16.5
H₂ O	12.17 ml	10.08 ml	7.46 ml
Heavy gel buffer	10 ml	10 ml	10 ml
Gel A/B	6.75 ml	8.78 ml	11.14 ml
Gel A	750 µl	975 µl	1.24 ml
TEMED	15 µl	15 µl	15 µl
10% APS	150 µl	150 µl	150 µl

	Acrylamide concentration of stacking gel (%) n
Stock solution	5 (lower)	5 (upper)	10 (lower)	10 (upper)
H₂ O	5.37 ml	5.25 ml	4.12 ml	4 ml
Light gel buffer	3.33 ml		3.33 ml
Native light gel buffer		3.33 ml		3.33 ml
20% SDS		100 µl		100 µl
Gel A/B	1.125 ml	1.125 ml	2.25 ml	2.25 ml
Gel A	125 µl	125 µl	250 µl	250 µl
TEMED	5 µl	7 µl	5 µl	7 µl
10% APS	50 µl	70 µl	50 µl	70 µl
Reagents used in Gels

Tricine‐SDS‐buffer, 3× (glycerol)
90.86 g Tris (3 M final) 0.375 g SDS (0.15% final) 75 g glycerol (30% final) Dissolve carefully in 210 ml water (final volume) and 5 ml of 30% HCl. Adjust to pH 7.0 with 30% HCl. Add water to 250 ml total volume. Filter the solution through a 0.45‐µm filter and store up to 1 month at room temperature.

Tricine‐SDS‐buffer, 3×
90.86 g Tris (3 M final) 0.375 g SDS (0.15% final) Dissolve carefully in 210 ml water (final volume) and 5 ml of 30% HCl. Adjust to pH 7.0 with 30% HCl. Add water to 250 ml total volume. Filter the solution through a 0.45‐µm filter and store up to 1 month at room temperature.

^m The recipes produce 30 ml of each separating gel solution, which are sufficient for a single gel with 1.5‐mm × 14‐cm × 16‐cm dimensions (Hoefer). When using Bio‐Rad equipment (1.5‐mm × 16‐cm × 16‐cm) or other, the respective volumes must be adapted adequately. The resulting 10 ml of each stacking gel solution are sufficient for the respective stacking gels of two different gels.

ⁿ The content of the crosslinker N,N′ ‐methylene bis‐acrylamide is 3% in each gel.

Use Milli‐Q‐purified water or equivalent in all recipes and protocol steps.

Support Protocol 1: Removal of Coomassie Dye from Electroeluted Proteins

Materials

Sephacryl S‐100 HR (GE Healthcare)
Chromatography buffer (see recipe ), 4°C
Eluted protein sample
Chromatography columns (1‐cm i.d., 30‐ to 60‐cm height) with column extension (packing funnel), adaptors, and buffer reservoir
Low pressure chromatography system 500‐µl sample loop, e.g., GE Healthcare Äkta prime or Bio‐Rad BioLogic
Dual wavelength detector, optional but desirable

Additional reagents and equipment for gel filtration (see also unit 8.3 )

NOTE: Perform all steps at 4°C.

Support Protocol 2: Solubilization of Biological Membranes for Native Gel Electrophoresis

Materials

Sample containing biological membranes
Solubilization buffer (see reciperecipes )
Liquid nitrogen
Detergent stock solutions (see reciperecipes )
Sample buffer (see reciperecipes )
80% (v/v) acetone
BN‐ and/or CN‐PAGE gels

Centrifuge for test tubes
Test tubes
Tiny spatula
10‐ml volumetric flask
Funnel and analytical filter paper (at least 10‐ml capacity)
Absorption spectrometer

Support Protocol 3: Construction of the H‐Shaped Elution Device and the Electroelution Chamber

Materials

ACRIFIX 192 adhesive glue (EVONIK Industries)
Plexiglas plates (3‐, 4‐, 6‐, and 10–mm thicknesses)
Plexiglas rod (1‐cm diameter)
Plexiglas tubes: 1.5‐cm o.d. (2‐mm wall thickness); 2‐cm o.d. (3‐mm wall thickness); 3‐cm o.d. (5‐mm wall thickness); 2.5‐cm o.d. (6‐mm wall thickness); 3.5‐cm o.d. (8‐mm wall thickness)
Electrode connections
Platinum wire
Plastic screw with 2.5‐mm diameter thread, ∼1‐cm long
Teflon washers, 1.5‐mm thick (1.9‐cm o.d., 1.3‐cm i.d. and 2.6‐cm o.d., 1.9‐cm i.d.)
Rubber gaskets, 1‐mm thick (1.8‐cm and 2.5‐cm diameter)

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Figures

Figure 19.18.1 Flowchart of analysis of protein‐protein interactions by BN‐ and CN‐PAGE.

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Figure 19.18.2 Setup for preparing native gradient gels.

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Figure 19.18.3 Setup of the electroelution device. The inset shows the assembly of the vertical tubes of the eluter.

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Figure 19.18.4 Details for constructing the electroelution device. (A ) A side view of the electrophoretic chamber. (B ) Top view of this chamber. (C ) The dimensions of the H‐shaped device.

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Figure 19.18.5 Separation of protein complexes from digitonin‐solubilized bovine heart mitochondria (isolated from fresh tissue) in the mass range of ∼100 kDa to ∼3 MDa by a BN‐PAGE (4% to 13%) in the first dimension (upper panel, gel strip). The subunits of the protein complexes retained in the first dimension are separated by a second dimension 16.5% tricine‐SDS‐PAGE (lower panel) migrating in a vertical row under the positions of the respective protein complexes. Under these conditions, high amounts of respiratory supercomplexes as well as ATP synthase dimers and higher oligomers are preserved. Besides the ATP synthases (V_1‐4 ) the individual respiratory complexes I‐IV as well as the respiratory supercomplexes I₁ IV₁ and I₁ III₂ IV_0‐4 are indicated.

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Figure 19.18.6 Separation of solubilized protein complexes from cyanobacterial membranes by (A ) BN‐PAGE and (B ) CN‐PAGE (each 3% to 12%). The membranes were solubilized with digitonin (lanes 1 to 3) or DDM (lanes 4, 6, and 7) at a detergent/chlorophyll ratio of 5 g/g (lanes 1 and 4), 10 g/g (lanes 2 and 6), and 20 g/g (lanes 3 and 7). Lane 5 was loaded with a commercially available standard of soluble proteins (not used for mass calibration, see comments in ). Some of the bands from the soluble protein standard are indicated on the right side.

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Literature Cited

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Detection and Analysis of Protein‐Protein Interactions of Organellar and Prokaryotic Proteomes by Blue Native and Colorless Native Gel Electrophoresis

Abstract

Table of Contents

Materials

Basic Protocol 1: Standard Native Gels for High‐Resolution Separation of Protein Complexes

Alternate Protocol 1: Mini‐Native Gels for Fast Separation of Protein Complexes

Alternate Protocol 2: Preparative Native Gels for Isolation of Up to Milligram Amounts of Protein Complexes

Basic Protocol 2: Electroelution for Recovery of Proteins from Native Polyacrylamide Gels

Basic Protocol 3: Two‐Dimensional Blue‐Native Polyacrylamide Gel Electrophoresis (BN‐PAGE) to Separate Constituting Subcomplexes of Protein Complexes Retained in First‐Dimension Native Gels

Basic Protocol 4: Tricine‐SDS‐PAGE for Separation of Protein Complex Subunits in Second or Third Dimension

Support Protocol 1: Removal of Coomassie Dye from Electroeluted Proteins

Support Protocol 2: Solubilization of Biological Membranes for Native Gel Electrophoresis

Support Protocol 3: Construction of the H‐Shaped Elution Device and the Electroelution Chamber

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