3.3.3 Size Exclusion
Chromatography (SEC)
Analysis
1. Equilibrate a Superdex™ 200 Increase 5/150 GL column
(GE Healthcare) with a flow rate of 0.1 mL/min at 4
C with
3 column volumes (CV) of eluent buffer corresponding to the
buffer of the membrane protein to be analyzed (see Notes 12
and 13).
2. Inject the membrane protein sample onto the column
(volume 50 μL).
3. Elute the protein at 4
C with 1.5 CV of eluent buffer at
0.1 mL/min.
4. The elution fractions can be analyzed by SDS-PAGE and Western blot to confirm the presence of the membrane protein.
4 Notes
1. As the cell lysate will warm up, due to the high energy mixing
with glass beads, make sure to use an ice-water cooling jacket
with the Bead Beater homogenizer to control the temperature
during the cell disruption. The 2 min cool down step between
two cell lysis cycles is critical to avoid protein denaturation.
2. 2Â stock solutions of each detergent or combination of detergents are prepared in 1Â PBS, 1Â PIC at !20-fold the Critical
Micelle Concentration (CMC) in order to reach a final concentration of !10 CMC in the solubilization tests.
3. Keep one well with no membrane that will serve as negative
control and one well with 2% SDS that will serve as positive
control. An example of 96-well plate solubilization conditions
is described in [7].
4. The membrane should not extend beyond the edge of the
gasket after the Bio-Dot apparatus is assembled.
5. Use a diagonal crossing pattern when tightening the screws to
ensure uniform application of pressure on the membrane
surface.
6. Tightening the four screws while vacuum is applied ensures a
tight sealing, preventing leaking between the wells and contamination between slots.
7. To stop the vacuum, set the flow valve to a position that does
not expose the apparatus to air. That will maintain a slight
depression in the Bio-Dot apparatus preventing leaking
between the wells, while the vacuum pump is off.
8. This step will drastically improve the membrane protein retention on the GF/B filter.
9. The orthovanadate inhibits ATPase activity, enabling to determine the nonspecific inorganic phosphate background.
266
Vincent Corvest and Anass Jawhari
Chromatography (SEC)
Analysis
1. Equilibrate a Superdex™ 200 Increase 5/150 GL column
(GE Healthcare) with a flow rate of 0.1 mL/min at 4
C with
3 column volumes (CV) of eluent buffer corresponding to the
buffer of the membrane protein to be analyzed (see Notes 12
and 13).
2. Inject the membrane protein sample onto the column
(volume 50 μL).
3. Elute the protein at 4
C with 1.5 CV of eluent buffer at
0.1 mL/min.
4. The elution fractions can be analyzed by SDS-PAGE and Western blot to confirm the presence of the membrane protein.
4 Notes
1. As the cell lysate will warm up, due to the high energy mixing
with glass beads, make sure to use an ice-water cooling jacket
with the Bead Beater homogenizer to control the temperature
during the cell disruption. The 2 min cool down step between
two cell lysis cycles is critical to avoid protein denaturation.
2. 2Â stock solutions of each detergent or combination of detergents are prepared in 1Â PBS, 1Â PIC at !20-fold the Critical
Micelle Concentration (CMC) in order to reach a final concentration of !10 CMC in the solubilization tests.
3. Keep one well with no membrane that will serve as negative
control and one well with 2% SDS that will serve as positive
control. An example of 96-well plate solubilization conditions
is described in [7].
4. The membrane should not extend beyond the edge of the
gasket after the Bio-Dot apparatus is assembled.
5. Use a diagonal crossing pattern when tightening the screws to
ensure uniform application of pressure on the membrane
surface.
6. Tightening the four screws while vacuum is applied ensures a
tight sealing, preventing leaking between the wells and contamination between slots.
7. To stop the vacuum, set the flow valve to a position that does
not expose the apparatus to air. That will maintain a slight
depression in the Bio-Dot apparatus preventing leaking
between the wells, while the vacuum pump is off.
8. This step will drastically improve the membrane protein retention on the GF/B filter.
9. The orthovanadate inhibits ATPase activity, enabling to determine the nonspecific inorganic phosphate background.
266
Vincent Corvest and Anass Jawhari