8. Glacial acetic acid.
9. Triethylamine.
10. 100% acetone, stored at À20
C.
11. Dialysis tubing, 3.5 kDa MWCO.
12. Dialysis clips and foam float.
13. 10 mM ammonium bicarbonate, NH 4 HCO 3 (see Subheading
2.2, item 4).
14. Dialysis tank (7 L).
15. 2% w/v SDS in PBS.
2.8 Guanidine–HCL
Extraction
of Hydrophobic
Proteins (See Note 20)
1. Soluble/insoluble protein sample (such as cell wall or membrane obtained in Subheading 3.6).
2. 6 M Guanidine–HCL, freshly prepared.
3. Pyrogen-free, 0.2μm filtered water.
4. Oakridge centrifuge tubes (polypropylene or Teflon), 50 mL,
sterile.
5. Micro stir bars.
6. High-speed centrifuge and rotors.
7. Dialysis tubing, 3.5 kDa MWCO.
8. Dialysis clips and foam float.
9. 10 mM ammonium bicarbonate (see Subheading 2.2, item 4).
10. Dialysis tank (7 L).
2.9 8 M Urea
Extraction
of Hydrophobic
Proteins (See Note 20)
1. Soluble/insoluble protein sample (such as cell wall or membrane obtained in Subheading 3.6).
2. 8 M Urea: Urea should be deionized prior to use to remove
reactive cyanate ions. 8 M solutions of urea may be bulk deionized using an ion-exchange resin. Add AG-501-X8 (Bio-Rad,
Hercules, CA) at 5 g/100 mL, stir the solution for 2 h at RT
(22
C), and filter the solution to recover deionized 8 M urea.
3. Pyrogen-free, 0.2μm filtered water.
4. Oakridge centrifuge tubes (polypropylene or Teflon), 50 mL,
sterile.
5. Micro stir bars.
6. High-speed centrifuge and rotors.
7. Dialysis tubing, 3.5 kDa MWCO.
8. Dialysis clips and foam float.
9. 10 mM ammonium bicarbonate (see Subheading 2.2, item 4).
10. Dialysis tank (7 L).
82
Megan Lucas et al.
9. Triethylamine.
10. 100% acetone, stored at À20
C.
11. Dialysis tubing, 3.5 kDa MWCO.
12. Dialysis clips and foam float.
13. 10 mM ammonium bicarbonate, NH 4 HCO 3 (see Subheading
2.2, item 4).
14. Dialysis tank (7 L).
15. 2% w/v SDS in PBS.
2.8 Guanidine–HCL
Extraction
of Hydrophobic
Proteins (See Note 20)
1. Soluble/insoluble protein sample (such as cell wall or membrane obtained in Subheading 3.6).
2. 6 M Guanidine–HCL, freshly prepared.
3. Pyrogen-free, 0.2μm filtered water.
4. Oakridge centrifuge tubes (polypropylene or Teflon), 50 mL,
sterile.
5. Micro stir bars.
6. High-speed centrifuge and rotors.
7. Dialysis tubing, 3.5 kDa MWCO.
8. Dialysis clips and foam float.
9. 10 mM ammonium bicarbonate (see Subheading 2.2, item 4).
10. Dialysis tank (7 L).
2.9 8 M Urea
Extraction
of Hydrophobic
Proteins (See Note 20)
1. Soluble/insoluble protein sample (such as cell wall or membrane obtained in Subheading 3.6).
2. 8 M Urea: Urea should be deionized prior to use to remove
reactive cyanate ions. 8 M solutions of urea may be bulk deionized using an ion-exchange resin. Add AG-501-X8 (Bio-Rad,
Hercules, CA) at 5 g/100 mL, stir the solution for 2 h at RT
(22
C), and filter the solution to recover deionized 8 M urea.
3. Pyrogen-free, 0.2μm filtered water.
4. Oakridge centrifuge tubes (polypropylene or Teflon), 50 mL,
sterile.
5. Micro stir bars.
6. High-speed centrifuge and rotors.
7. Dialysis tubing, 3.5 kDa MWCO.
8. Dialysis clips and foam float.
9. 10 mM ammonium bicarbonate (see Subheading 2.2, item 4).
10. Dialysis tank (7 L).
82
Megan Lucas et al.