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

Frank Krause1, Holger Seelert1

1 Technische Universität Darmstadt, Darmstadt, Germany
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.18
DOI:  10.1002/0471140864.ps1918s54
Online Posting Date:  November, 2008
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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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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
     
 
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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.

     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

  • 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
    H 2O 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
    H 2O 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
    H 2O 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.

     eThe 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.
     fThe content of the cross‐linker N,N′‐methylene bis‐acrylamide is 3% in each gel.
     gAll reagents and solutions in the protocol must be prepared with Milli‐Q‐purified water or equivalent.
     hFor 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
    H 2O 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
    H 2O 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
    H 2O 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.

     JThe 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.
     kThe content of the crosslinker N,N′‐methylene bis‐acrylamide is 3% in each gel.
     lAll 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
    H 2O 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)
    H 2O 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.

     mThe 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.
     nThe 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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Literature Cited

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