2.3 ATPase
Activity Assay
1. Inorganic phosphate (Pi) standard stock solution: 10 mM
Na 2 HPO 4 , pH 8.0.
2. Adenosine 5
0 -triphosphate (ATP).
3. Sodium orthovanadate (Na 3 VO 4 ) heated at 95
C.
4. Tris-buffered saline (1Â TBS): 50 mM Tris–HCl, 50 mM
NaCl, pH 8.0.
5. Reaction buffer: 50 mM Tris–HCl, 50 mM NaCl, 10 mM
MgCl 2 , 6 mM NaN 3 , pH 8.0.
6. Revelation buffer (to be prepared just before use): 45 mM
ascorbic acid, 17.5 mM ammonium molybdate, 15 mM zinc
acetate, pH 5.0.
2.4 Clear-NativePAGE
1. 5Â native loading buffer: 250 mM Tris–HCl, 50% glycerol,
0.25% bromophenol blue, pH 8.0.
2. CN-PAGE anode buffer: 25 mM imidazole, pH 8.0.
3. CN-PAGE cathode buffer: 50 mM tricine, 0.05%
Na-deoxycholate, 0.01% n-dodecyl β-D-maltoside (DDM,
Carl Roth), 7.5 mM imidazole, pH 8.0.
2.5 Glutaraldehyde
Cross-Linking Assay
1. Glutaraldehyde (Carl Roth).
2. 5Â SDS-DTT loading buffer: 250 mM Tris–HCl, 10% sodium
dodecyl sulfate (SDS), 0.5 M dithiothreitol (DTT), 50% glycerol, 0.25% bromophenol blue, pH 8.0.
3 Methods
3.1 Screening of
Membrane Proteins
Solubilization
Here we describe a 96-well microplate solubilization screening for
membrane proteins using the micro-ultracentrifugation to separate
soluble from insoluble fractions. For limited microultracentrifugation capacity, an alternative using the biotinylated
membranes solubilization and separation method is described
in [7].
3.1.1 Cell Lysis and
Membrane Fractionation
1. Resuspend dry cell pellet at 2 mL/g with 1Â PBS supplemented with 1Â PIC.
2. Lyse cells on ice using a Bead Beater homogenizer with
0.1 mm diameter glass beads (0.5 mm for yeast) with five cycles
of 30 s lysis followed by 2 min cool down (see Note 1).
3. Centrifuge at increasing speeds at 4
C to collect different
membrane fractions: 1000 Â g for 5 min (whole cell and cell
debris), 15,000 Â g for 30 min (internal membranes, ER,
mitochondria, etc.) and 100,000 Â g for 45 min (plasma
membrane).
Solubilization and Stabilization of Membrane Proteins
261
Activity Assay
1. Inorganic phosphate (Pi) standard stock solution: 10 mM
Na 2 HPO 4 , pH 8.0.
2. Adenosine 5
0 -triphosphate (ATP).
3. Sodium orthovanadate (Na 3 VO 4 ) heated at 95
C.
4. Tris-buffered saline (1Â TBS): 50 mM Tris–HCl, 50 mM
NaCl, pH 8.0.
5. Reaction buffer: 50 mM Tris–HCl, 50 mM NaCl, 10 mM
MgCl 2 , 6 mM NaN 3 , pH 8.0.
6. Revelation buffer (to be prepared just before use): 45 mM
ascorbic acid, 17.5 mM ammonium molybdate, 15 mM zinc
acetate, pH 5.0.
2.4 Clear-NativePAGE
1. 5Â native loading buffer: 250 mM Tris–HCl, 50% glycerol,
0.25% bromophenol blue, pH 8.0.
2. CN-PAGE anode buffer: 25 mM imidazole, pH 8.0.
3. CN-PAGE cathode buffer: 50 mM tricine, 0.05%
Na-deoxycholate, 0.01% n-dodecyl β-D-maltoside (DDM,
Carl Roth), 7.5 mM imidazole, pH 8.0.
2.5 Glutaraldehyde
Cross-Linking Assay
1. Glutaraldehyde (Carl Roth).
2. 5Â SDS-DTT loading buffer: 250 mM Tris–HCl, 10% sodium
dodecyl sulfate (SDS), 0.5 M dithiothreitol (DTT), 50% glycerol, 0.25% bromophenol blue, pH 8.0.
3 Methods
3.1 Screening of
Membrane Proteins
Solubilization
Here we describe a 96-well microplate solubilization screening for
membrane proteins using the micro-ultracentrifugation to separate
soluble from insoluble fractions. For limited microultracentrifugation capacity, an alternative using the biotinylated
membranes solubilization and separation method is described
in [7].
3.1.1 Cell Lysis and
Membrane Fractionation
1. Resuspend dry cell pellet at 2 mL/g with 1Â PBS supplemented with 1Â PIC.
2. Lyse cells on ice using a Bead Beater homogenizer with
0.1 mm diameter glass beads (0.5 mm for yeast) with five cycles
of 30 s lysis followed by 2 min cool down (see Note 1).
3. Centrifuge at increasing speeds at 4
C to collect different
membrane fractions: 1000 Â g for 5 min (whole cell and cell
debris), 15,000 Â g for 30 min (internal membranes, ER,
mitochondria, etc.) and 100,000 Â g for 45 min (plasma
membrane).
Solubilization and Stabilization of Membrane Proteins
261