4. Add 40 μL of reaction buffer to each well.
5. Add 2 μL of 150 mM ATP to each protein sample well, and
2 μL 1Â TBS to each Pi standard well.
6. Add 2 μL of 1Â TBS to series #1 well, and 2 μL of hot 6 mM
orthovanadate to series #2 well (see Note 9).
7. Incubate at 37
C for 60 min under gentle agitation.
8. Add 30 μL 10% SDS to stop the reaction.
9. Incubate at room temperature for 10 min under gentle
agitation.
10. Add 180 μL of revelation buffer (freshly prepared).
11. Incubate at 37
C for 20 min under gentle agitation.
12. Measure the absorbance at 620 nm.
13. Calculate for each protein sample the total ATPase activity
(series #1) and the nonspecific ATPase activity (series #2)
using the corresponding standard curve.
14. Determine the specific ATPase activity for each protein sample
by subtracting the nonspecific signal (series #2) from the total
signal (series #1).
3.3 Behavior of
Membrane Protein in
Solution
3.3.1 Clear-Native-PAGE
1. Add appropriate volume of 5Â native loading buffer to the
membrane protein samples leading to 1Â final concentration
(see Note 10).
2. Load the sample in a 4–15% Mini-PROTEAN
® TGX™ precast
protein gel (Bio-Rad) and run the gel for 70 min at 4
C using
CN-PAGE anode and cathode buffers.
3. Detect the protein by Coomassie staining or Western blot for
an immunodetection (see Note 11).
3.3.2 Glutaraldehyde
Cross-Linking Assay
1. Prepare a range of glutaraldehyde stock solutions in water: 0%,
0.0625%, 0.125%, 0.25%, 0.5%.
2. Aliquot 10 μL of purified membrane protein into five
microtubes.
3. Add 2.5 μL of different glutaraldehyde stock solutions in order
to reach 0%, 0.0125%, 0.025%, 0.05%, and 0.1% final
concentrations.
4. Incubate the samples on ice for 2 h.
5. Stop the crosslinking reaction by adding 3.5 μL of 5Â
SDS-DTT loading buffer.
6. Analyze the samples by SDS-PAGE and Western blot.
Solubilization and Stabilization of Membrane Proteins
265
5. Add 2 μL of 150 mM ATP to each protein sample well, and
2 μL 1Â TBS to each Pi standard well.
6. Add 2 μL of 1Â TBS to series #1 well, and 2 μL of hot 6 mM
orthovanadate to series #2 well (see Note 9).
7. Incubate at 37
C for 60 min under gentle agitation.
8. Add 30 μL 10% SDS to stop the reaction.
9. Incubate at room temperature for 10 min under gentle
agitation.
10. Add 180 μL of revelation buffer (freshly prepared).
11. Incubate at 37
C for 20 min under gentle agitation.
12. Measure the absorbance at 620 nm.
13. Calculate for each protein sample the total ATPase activity
(series #1) and the nonspecific ATPase activity (series #2)
using the corresponding standard curve.
14. Determine the specific ATPase activity for each protein sample
by subtracting the nonspecific signal (series #2) from the total
signal (series #1).
3.3 Behavior of
Membrane Protein in
Solution
3.3.1 Clear-Native-PAGE
1. Add appropriate volume of 5Â native loading buffer to the
membrane protein samples leading to 1Â final concentration
(see Note 10).
2. Load the sample in a 4–15% Mini-PROTEAN
® TGX™ precast
protein gel (Bio-Rad) and run the gel for 70 min at 4
C using
CN-PAGE anode and cathode buffers.
3. Detect the protein by Coomassie staining or Western blot for
an immunodetection (see Note 11).
3.3.2 Glutaraldehyde
Cross-Linking Assay
1. Prepare a range of glutaraldehyde stock solutions in water: 0%,
0.0625%, 0.125%, 0.25%, 0.5%.
2. Aliquot 10 μL of purified membrane protein into five
microtubes.
3. Add 2.5 μL of different glutaraldehyde stock solutions in order
to reach 0%, 0.0125%, 0.025%, 0.05%, and 0.1% final
concentrations.
4. Incubate the samples on ice for 2 h.
5. Stop the crosslinking reaction by adding 3.5 μL of 5Â
SDS-DTT loading buffer.
6. Analyze the samples by SDS-PAGE and Western blot.
Solubilization and Stabilization of Membrane Proteins
265