4. Resuspend membrane fractions at 0.5 mL/g of initial dry cell
pellet with 1Â PBS, 1Â PIC, 10% (v/v) glycerol.
5. Quantify total membrane proteins of each fraction using the
micro BCA protein assay kit (Thermo Scientific) and determine
the protein of interest localization by SDS-PAGE and
Western blot.
6. Aliquot, flash-freeze in liquid nitrogen and store membrane
fractions at À80
C until use.
3.1.2 96-Well Microplate
Solubilization
1. Prepare 1 mL of 2Â stock solutions in 1Â PBS, 1Â PIC of each
detergent or combination of detergents to be tested in a
96-deep well plate (sufficient for 20 tests of 96-well microplate
solubilization) (see Note 2).
2. Dilute the membrane fraction containing the protein of interest at 2 mg/mL in 1Â PBS, 1Â PIC.
3. Aliquot 50 μL diluted membranes into a 96-well microplate
(see Note 3).
4. Using a multi-channel pipet transfer 50 μL of detergent solution from the 96-deep well 2Â stock solutions plate to the
96-well solubilization microplate containing the membranes.
5. Incubate the 96-well solubilization microplate at 4
C for 2 h
under agitation.
6. Transfer the 100 μL solubilisates into 0.5 mL microultracentrifuge tubes and ultracentrifuge at 150,000 Â g for
15 min at 4
C.
7. Transfer the supernatants (soluble fractions) in a 96-well
microplate at 4
C.
3.1.3 Dot-Blot
1. Soak the nitrocellulose membrane in 1Â PBS for 10 min at
room temperature.
2. Assemble the Bio-Dot
® microfiltration apparatus (Bio-Rad).
3. Lay the membrane on the sealing gasket so that it covers all of
the holes and remove any air bubbles trapped between the
membrane and the sealing gasket (see Note 4).
4. Place the sample template on top of the membrane and fingertighten the four screws (see Note 5).
5. Attach a vacuum pump to the flow valve with a waste trap set up
and positioned between the vacuum outlet and the flow valve.
Turn on the vacuum pump (À20 kPa pressure) and set the flow
valve to apply vacuum to the apparatus (flow valve position 2).
6. Repeat the four screws tightening process (see Note 6).
7. Set the flow valve to stop vacuum (flow valve position 1) and
turn off the vacuum pump (see Note 7).
262
Vincent Corvest and Anass Jawhari
pellet with 1Â PBS, 1Â PIC, 10% (v/v) glycerol.
5. Quantify total membrane proteins of each fraction using the
micro BCA protein assay kit (Thermo Scientific) and determine
the protein of interest localization by SDS-PAGE and
Western blot.
6. Aliquot, flash-freeze in liquid nitrogen and store membrane
fractions at À80
C until use.
3.1.2 96-Well Microplate
Solubilization
1. Prepare 1 mL of 2Â stock solutions in 1Â PBS, 1Â PIC of each
detergent or combination of detergents to be tested in a
96-deep well plate (sufficient for 20 tests of 96-well microplate
solubilization) (see Note 2).
2. Dilute the membrane fraction containing the protein of interest at 2 mg/mL in 1Â PBS, 1Â PIC.
3. Aliquot 50 μL diluted membranes into a 96-well microplate
(see Note 3).
4. Using a multi-channel pipet transfer 50 μL of detergent solution from the 96-deep well 2Â stock solutions plate to the
96-well solubilization microplate containing the membranes.
5. Incubate the 96-well solubilization microplate at 4
C for 2 h
under agitation.
6. Transfer the 100 μL solubilisates into 0.5 mL microultracentrifuge tubes and ultracentrifuge at 150,000 Â g for
15 min at 4
C.
7. Transfer the supernatants (soluble fractions) in a 96-well
microplate at 4
C.
3.1.3 Dot-Blot
1. Soak the nitrocellulose membrane in 1Â PBS for 10 min at
room temperature.
2. Assemble the Bio-Dot
® microfiltration apparatus (Bio-Rad).
3. Lay the membrane on the sealing gasket so that it covers all of
the holes and remove any air bubbles trapped between the
membrane and the sealing gasket (see Note 4).
4. Place the sample template on top of the membrane and fingertighten the four screws (see Note 5).
5. Attach a vacuum pump to the flow valve with a waste trap set up
and positioned between the vacuum outlet and the flow valve.
Turn on the vacuum pump (À20 kPa pressure) and set the flow
valve to apply vacuum to the apparatus (flow valve position 2).
6. Repeat the four screws tightening process (see Note 6).
7. Set the flow valve to stop vacuum (flow valve position 1) and
turn off the vacuum pump (see Note 7).
262
Vincent Corvest and Anass Jawhari