8. Apply 100 μL 1Â PBS to all 96 wells.
9. Turn on the vacuum pump and set the flow valve to position
2. Gently remove the buffer from the wells. As soon as the
buffer drains from all the wells, set the flow valve to position
1 and turn off the pump.
10. Fill the appropriate wells with 50 μL of supernatant from
solubilization and ensure that there are no air bubbles in the
wells.
11. Allow the samples to filter through the membrane by gravity
for 5 min.
12. Turn on the vacuum pump, set the flow valve to position 2, and
allow complete sample filtration with vacuum.
13. With vacuum applied, loosen the four screws and remove the
sample template.
14. Set the flow valve to position 1, turn off the vacuum pump, and
remove the nitrocellulose membrane.
15. Block the nitrocellulose membrane with 30 mL blocking
buffer and immunodetect the protein of interest using a specific antibody.
16. Confirm the optimal conditions of solubilization by
SDS-PAGE and Western blot analysis and Western blot–based
thermostability assay.
3.2 Membrane
Protein Stability
Assessment
3.2.1 Western Blot–
Based
Thermostability Assay
The Western blot–based thermostability assay is adapted from [8].
1. Solubilize the protein of interest in each detergent condition to
be evaluated and in a final volume of 700 μL.
2. Aliquot 50 μL of solubilisate into 14 PCR microtubes.
3. Using a thermocycler, submit the PCR microtubes to a gradient of temperature from 25
C to 80
C for 30 min.
4. Immediately transfer to fresh microtubes and centrifuge at
20,000 Â g for 40 min at 4
C.
5. Analyze the supernatants by SDS-PAGE and Western blot
using an antibody raised against the protein of interest.
6. Quantify the intensity of the immunodetection signal,
corresponding to the protein of interest, plot as function of
temperature, and fit to the Boltzmann equation to determine
the T m .
3.2.2 Radioligand
Binding Assay
This protocol is developed for A 2A receptor and is adapted from
[9]. A single protein concentration and a single radioligand concentration are used to compare the different protein preparations.
This assay is performed in triplicate for each protein sample.
Solubilization and Stabilization of Membrane Proteins
263
9. Turn on the vacuum pump and set the flow valve to position
2. Gently remove the buffer from the wells. As soon as the
buffer drains from all the wells, set the flow valve to position
1 and turn off the pump.
10. Fill the appropriate wells with 50 μL of supernatant from
solubilization and ensure that there are no air bubbles in the
wells.
11. Allow the samples to filter through the membrane by gravity
for 5 min.
12. Turn on the vacuum pump, set the flow valve to position 2, and
allow complete sample filtration with vacuum.
13. With vacuum applied, loosen the four screws and remove the
sample template.
14. Set the flow valve to position 1, turn off the vacuum pump, and
remove the nitrocellulose membrane.
15. Block the nitrocellulose membrane with 30 mL blocking
buffer and immunodetect the protein of interest using a specific antibody.
16. Confirm the optimal conditions of solubilization by
SDS-PAGE and Western blot analysis and Western blot–based
thermostability assay.
3.2 Membrane
Protein Stability
Assessment
3.2.1 Western Blot–
Based
Thermostability Assay
The Western blot–based thermostability assay is adapted from [8].
1. Solubilize the protein of interest in each detergent condition to
be evaluated and in a final volume of 700 μL.
2. Aliquot 50 μL of solubilisate into 14 PCR microtubes.
3. Using a thermocycler, submit the PCR microtubes to a gradient of temperature from 25
C to 80
C for 30 min.
4. Immediately transfer to fresh microtubes and centrifuge at
20,000 Â g for 40 min at 4
C.
5. Analyze the supernatants by SDS-PAGE and Western blot
using an antibody raised against the protein of interest.
6. Quantify the intensity of the immunodetection signal,
corresponding to the protein of interest, plot as function of
temperature, and fit to the Boltzmann equation to determine
the T m .
3.2.2 Radioligand
Binding Assay
This protocol is developed for A 2A receptor and is adapted from
[9]. A single protein concentration and a single radioligand concentration are used to compare the different protein preparations.
This assay is performed in triplicate for each protein sample.
Solubilization and Stabilization of Membrane Proteins
263