1. Prepare the purified membrane proteins at 24 μg/mL in their
own buffer.
2. Using a 96-well microplate with a U-bottom, aliquot 50 μL of
protein sample at 24 μg/mL into two wells (well #1 for the
total signal and well #2 for the nonspecific signal).
3. Add 10 μL of radioligand solution to the well #1 (total signal)
and 10 μL of nonspecific ligand solution to the well #2 (nonspecific signal).
4. Mix by pipetting up and down, and incubate at 4
C for 2 h.
5. Add 60 μL of γ-globulin solution and 120 μL PEG6000 solution to each well (see Note 8).
6. Mix by pipetting up and down, and incubate at 4
C for
15 min.
7. Using the FilterMate Harvester:
(a) Rinse twice the PEI-coated GF/B filter 96-well microplate with 200 μL of wash buffer.
(b) Transfer the protein samples to the PEI-coated GF/B
filter 96-well microplate.
(c) Wash four times the PEI-coated GF/B filter 96-well
microplate with 200 μL of wash buffer.
(d) Dry the PEI-coated GF/B filter 96-well microplate for
2 min under vacuum.
8. Seal the bottom of PEI-coated GF/B filter 96-well microplate
with the adhesive BackSeal.
9. Add 25 μL of scintillation reagent to each well.
10. Seal the top of PEI-coated GF/B filter 96-well microplate with
the adhesive TopSeal.
11. Incubate the plate for 1 h at room temperature.
12. Top count the CPM for 5 min per well using a microplate
counter. Determine the specific signal by subtracting the nonspecific signal (well #2) from the total signal (well #1).
3.2.3 ATPase
Activity Assay
This protocol is developed for BmrA transporter and is adapted
from [11].
1. Prepare a range of 20 μL Pi standards at 0 mM, 2 mM, 4 mM,
and 8 mM in 1Â TBS from the Pi standard stock solution.
2. Prepare the membrane protein samples at 0.5 mg/mL in their
own buffer.
3. Using a 96-well microplate with a transparent flat-bottom,
prepare 2 series of 10 μL for each Pi standards and protein
samples (series #1 for total signal, series #2 for nonspecific
signal).
264
Vincent Corvest and Anass Jawhari
own buffer.
2. Using a 96-well microplate with a U-bottom, aliquot 50 μL of
protein sample at 24 μg/mL into two wells (well #1 for the
total signal and well #2 for the nonspecific signal).
3. Add 10 μL of radioligand solution to the well #1 (total signal)
and 10 μL of nonspecific ligand solution to the well #2 (nonspecific signal).
4. Mix by pipetting up and down, and incubate at 4
C for 2 h.
5. Add 60 μL of γ-globulin solution and 120 μL PEG6000 solution to each well (see Note 8).
6. Mix by pipetting up and down, and incubate at 4
C for
15 min.
7. Using the FilterMate Harvester:
(a) Rinse twice the PEI-coated GF/B filter 96-well microplate with 200 μL of wash buffer.
(b) Transfer the protein samples to the PEI-coated GF/B
filter 96-well microplate.
(c) Wash four times the PEI-coated GF/B filter 96-well
microplate with 200 μL of wash buffer.
(d) Dry the PEI-coated GF/B filter 96-well microplate for
2 min under vacuum.
8. Seal the bottom of PEI-coated GF/B filter 96-well microplate
with the adhesive BackSeal.
9. Add 25 μL of scintillation reagent to each well.
10. Seal the top of PEI-coated GF/B filter 96-well microplate with
the adhesive TopSeal.
11. Incubate the plate for 1 h at room temperature.
12. Top count the CPM for 5 min per well using a microplate
counter. Determine the specific signal by subtracting the nonspecific signal (well #2) from the total signal (well #1).
3.2.3 ATPase
Activity Assay
This protocol is developed for BmrA transporter and is adapted
from [11].
1. Prepare a range of 20 μL Pi standards at 0 mM, 2 mM, 4 mM,
and 8 mM in 1Â TBS from the Pi standard stock solution.
2. Prepare the membrane protein samples at 0.5 mg/mL in their
own buffer.
3. Using a 96-well microplate with a transparent flat-bottom,
prepare 2 series of 10 μL for each Pi standards and protein
samples (series #1 for total signal, series #2 for nonspecific
signal).
264
Vincent Corvest and Anass Jawhari