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Detection and Analysis of Protein‐Protein Interactions of Organellar and Prokaryotic Proteomes by Blue Native and Colorless Native Gel Electrophoresis
Publication Name:
Current Protocols in Protein Science
Unit Number:
Unit 19.18
DOI:
10.1002/0471140864.ps1918s54
Online Posting Date:
November, 2008 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
Table of Contents
- 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
Materials
Basic Protocol 1: Standard Native Gels for High-Resolution Separation of Protein Complexes
Materials
- Separating and sample gel solutions (Table
- Samples of soluble proteins or solubilized membrane proteins (see
- Native standard proteins (e.g., in the range of 100- to 1000-kDa)
- Sample buffer (see
- Anode and cathode buffers (see
- 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 19.18.1 Recipes for Standard Size Polyacrylamide Separating and Stacking Gels
Light Acrylamide Gel Solutions for Standard Size Gradient Gels Acrylamide concentration of light gel solution (%) Stock solution 3 3.5 4 4.5 5 6 Water 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 (%) Stock solution 11 13 14 16 18 20 Water 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 (%) Stock solution 3 3.5 4 4.5 5 Water 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.
aThe 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.bThe content of the cross-linker N,N¢-methylene bis-acrylamide is 3% in each gel.cUse Milli-Q-purified water or equivalent in all recipes and protocol steps.dFor 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
Additional Materials (also see Basic Protocol 1 )
- Separating and sample gel solutions (Table
- 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 bagsTable 19.18.2 Recipes for Polyacrylamide Separating and Stacking Gels
Light Acrylamide Gel Solutions for Gradient Mini Gels Acrylamide concentration of light gel solution (%) Stock solution 3 3.5 4 4.5 5 6 H 2 O4.11 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 (%) Stock solution 11 13 14 16 18 20 H 2 O2.72 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 (%) Stock solution 3% 3.5 4 4.5 5 H 2 O2.33 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 aThe 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.bThe content of the cross-linker N,N¢-methylene bis-acrylamide is 3% in each gel.cAll reagents and solutions in the protocol must be prepared with Milli-Q-purified water or equivalent.dFor 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
Additional Materials (also see Basic Protocol 1 )
- Separating and sample gel solutions (Table
- 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 19.18.3 Recipes for Polyacrylamide Separating and Stacking Gels
Light Acrylamide Gel Solutions for Preparative Gradient Gels Acrylamide concentration of light gel solution (%) Stock solution 3 3.5 4 4.5 5 H 2 O19.93 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 (%) Stock solution 7 9 11 12 13 15 H 2 O16.56 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 (%) Stock solution 3 3.5 4 4.5 5 H 2 O11.65 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 aThe 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.bThe content of the crosslinker N,N¢-methylene bis-acrylamide is 3% in each gel.cAll 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
- 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. (
- Elution tank for H-shaped eluter (see
- 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
- First dimension BN-gel containing the protein bands of sample
- Separating and sample gel solutions (Table
- Native standard proteins (e.g., in the range of 100 to 1000 kDa)
- Sample buffer (see
- 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
- 1% SDS or a 1% SDS/1% mercaptoethanol solution
- Separating and stacking gel solutions (Table
- Denatured standard proteins
- Tricine gel anode and cathode buffer (see
- 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 19.18.4 Recipes for Standard Size Tricine Polyacrylamide Separating and Stacking Gels
Acrylamide concentration of separating gel (%) Stock solution 10 13 16.5 H 2 O12.17 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 (%) Stock solution 5 (lower) 5 (upper) 10 (lower) 10 (upper) H 2 O5.37 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.
aThe 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.bThe content of the crosslinker N,N¢-methylene bis-acrylamide is 3% in each gel.cUse 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
- 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
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
- Liquid nitrogen
- Detergent stock solutions (see
- Sample buffer (see
- 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 19.18.1Flowchart of analysis of protein-protein interactions by BN- and CN-PAGE. -
Figure 19.18.2Setup for preparing native gradient gels. -
Figure 19.18.3Setup of the electroelution device. The inset shows the assembly of the vertical tubes of the eluter. -
Figure 19.18.4Details 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. -
Figure 19.18.5Separation 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 (V1-4 ) the individual respiratory complexes I-IV as well as the respiratory supercomplexes I1 IV1 and I1 III2 IV0-4 are indicated. -
Figure 19.18.6Separation 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
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