Human PGP-ATPase Assay
Summary | Materials
| Solutions | Assay
Procedure
ASSAY PROCEDURES
- ATPase Assay: User Method
Evaluation in human PGP (453228 [Old K228]) and/or Control (453200 [Old
K200]) Membranes
- ATPase Assay: Drug Concentration
- Dependence
- Analysis of ATPase Assay
Data
Note: To run one full 96-well plate, two vials of
membrane (Cat. No. 453228) would need to be purchased.
1. ATPase Assay:
User Method Evaluation in human PGP(453228 [Old K228]) and/or Control
(453200 [Old K200]) Membranes
This is a procedure for evaluating the ATPase assay in human PGP and/or
control membranes. See plate set-up, corresponding absorbance values,
and sample calculations below. This assay is a modification of the methods
of Sarkadi et al., (1992) J. Biol. Chem. 267:4854 and Druekes et al.,
(1995) Analyt. Biochem. 230:173.
| 1. |
Prepare KPO4 standards (0, 0.05, 0.15, 0.50, 1.0, 1.5,
2.0 and 2.5mM) in Tris-Mes buffer, and add 60uL of each standard (0,
3, 9, 30, 60, 90, 120 and 150nmoles) to the appropriate wells in rows
A-H, cols 1 and 2. |
| 2. |
Add 20ul of Tris-Mes buffer to rows A and B, cols 3-10. |
| 3. |
Prepare 60uM Verapamil in Tris-Mes buffer and add 20ul
to rows C and D, cols 3-10. |
| 4. |
Prepare 300uM sodium orthovanadate in Tris-Mes buffer
and add 20ul to rows E and F, cols 3-10. |
| 5. |
Prepare a solution containing: 60uM Verapamil and 300uM
sodium orthovanadate in Tris-Mes buffer, and add 20ul to rows G and
H, cols 3-10. |
| 6. |
Prepare membranes at 2ug/ul in Tris-Mes buffer for
both 453228 (Old K228) and 453200 (Old K200). Do not vortex membranes.
Mix by gentle inversion. |
| 7. |
Add 20ul of 2ug/ul 453228 (Old K228) membranes (40ug/well)
to rows A-H, cols 3-6. Add 20ul of 2ug/ul 453200 (Old K200) membranes
(40ug/well) to rows A-H, cols 7-10. |
| 8. |
Incubate plate at 37°C for 3-5 minutes. |
| 9. |
While plate is incubating, prepare MgATP in Tris-Mes
buffer (e.g. prepare 12mM MgATP if 4mM final concentration is desired). |
| 10. |
Remove plate from incubation. Add 20ul of MgATP to the
t=20 wells (rows A-H, cols 5,6,9,10) with a multichannel pipetter.
Place remaining MgATP on ice to use later. |
| 11. |
Incubate plate at 37°C for 20 min. While
plate is incubating, prepare 10% Ascorbic acid. |
| 12. |
Remove plate from incubation and add 30ul
of 10% SDS+Antifoam A to the t=20 wells (rows A-H, cols 5,6,9,10). |
| 13. |
Add 30ul of 10%SDS+Antifoam A to all other
wells in the plate (including standards). |
| 14. |
Add 20ul of the MgATP solution to the t=0
wells (rows A-H, cols 3,4,7,8). |
15. |
Prepare the detection reagent: Add 5mls of 35mM Ammonium
Molybdate in 15mM Zinc Acetate, to 20mls of the freshly prepared 10%
ascorbic acid. Invert to mix. |
| 16. |
Using a multichannel (12 channel) pipettor,
or one that delivers to the entire plate at once, add 200ul of the
detection reagent to every well in the plate, and incubate plate at
37°C, in the dark, for 20 min. |
| 17. |
Read the plate on a microplate spectrophotometer
between 630 and 850 nm. |
ATPase Assay : User Evaluation Method in PGP and Control
Membranes
Example plate set-up, absorbance values, and calculation of ATPase Activity
|
|
Example 96-well Plate Set- Up:
ATPase Activities in Human MDR1 (453228 (Old K228)) and Control
(453200 (Old K200)) Membranes
|
|
KPO4 Stds
|
453228 (Old K228) Membranes
|
K220 Membranes
|
| |
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
|
| |
|
|
T = 0
|
T = 20
|
T = 0
|
T = 20
|
|
A
|
0
|
0
|
BUFFER
|
|
B
|
3
|
3
|
BUFFER
|
|
C
|
9
|
9
|
VERAPAMIL
|
|
D
|
30
|
30
|
VERAPAMIL
|
|
E
|
60
|
60
|
ORTHOVANADATE
|
|
F
|
90
|
90
|
ORTHOVANADATE
|
|
G
|
120
|
120
|
VERAP + ORTHOVANADATE
|
|
H
|
150
|
150
|
VERAP + ORTHOVANADATE
|
|
Corresponding Absorbance Values From Plate
read at 800nm
(Biotek Power Wave 340 Spectrophotometer)
|
|
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
|
| A |
0.082
|
0.080
|
0.178
|
0.176
|
0.430
|
0.419
|
0.198
|
0.202
|
0.365
|
0.379
|
|
B
|
0.126
|
0.122
|
0.167
|
0.168
|
0.380
|
0.378
|
0.186
|
0.188
|
0.347
|
0.389
|
| C |
0.210
|
0.195
|
0.167
|
0.168
|
0.679
|
0.631
|
0.187
|
0.203
|
0.377
|
0.360
|
| D |
0.531
|
0.519
|
0.159
|
0.165
|
0.590
|
0.614
|
0.192
|
0.193
|
0.364
|
0.386
|
| E |
0.844
|
0.841
|
0.162
|
0.162
|
0.340
|
0.330
|
0.197
|
0.199
|
0.317
|
0.325
|
| F |
1.260
|
1.217
|
0.173
|
0.173
|
0.351
|
0.317
|
0.203
|
0.192
|
0.326
|
0.366
|
| G |
1.420
|
1.418
|
0.175
|
0.173
|
0.329
|
0.333
|
0.199
|
0.191
|
0.339
|
0.342
|
| H |
1.884
|
1.868
|
0.177
|
0.184
|
0.360
|
0.349
|
0.210
|
0.218
|
0.362
|
0.365
|
|
Human Pgp-ATPase Assay - Sample Calculation
Sheet
|
|
Phosphate Standards
|
Regression Statistics
|
|
nmoles
|
mean abs
|
|
|
0
|
0.081
|
b(0) = 0.1149
|
|
3
|
0.124
|
b(1) = 0.0117
|
|
9
|
0.203
|
r2 = 0.9932
|
|
30
|
0.525
|
|
|
60
|
0.843
|
|
|
90
|
1.239
|
(Absorbance read at 800nm)
|
|
120
|
1.419
|
|
|
150
|
1.876
|
|
|
CALCULATION OF ATPase ACTIVITIES
In 453228 (Old K228) (human Pgp) and 453200 (Old K200) (control)
Membranes
|
| |
mean absorbance
|
| |
453228 (Old K228) Membranes
|
453200 (Old K200) Membranes
|
| |
T=0
|
T=20
|
T=0
|
T=20
|
| Buffer |
0.172
|
0.402
|
0.194
|
0.370
|
| Verapamil |
0.165
|
0.629
|
0.194
|
0.372
|
| NaOV |
0.168
|
0.335
|
0.198
|
0.334
|
| Ver + NaOV |
0.177
|
0.343
|
0.205
|
0.350
|
| |
nmoles of phosphate released
|
| |
453228 (Old K228) Membranes
|
453200 (Old K200) Membranes
|
| |
T=0
|
T=20
|
T=0
|
T=20
|
| Buffer |
4.90
|
24.52
|
6.72
|
21.80
|
| Verapamil |
4.26
|
43.90
|
6.74
|
21.95
|
| NaOV |
4.50
|
18.77
|
7.08
|
18.68
|
| Ver + NaOV |
5.33
|
19.47
|
7.66
|
20.07
|
| |
nmoles of phosphate released
in 20 minutes
|
| |
(T=20 – T=0)
|
| |
453228 (Old K228) Membranes
|
453200 (Old K200) Membranes
|
| Buffer |
19.62
|
15.09
|
| Verapamil |
39.64
|
15.21
|
| NaOV |
14.27
|
11.60
|
| Ver + NaOV |
14.15
|
12.41
|
| |
vanadate-sensitive nmoles of phosphate released
|
| |
453228 (Old K228) Membranes
|
453200 (Old K200) Membranes
|
| Buffer - NaOV |
5.34
|
3.48
|
| Verapamil – Ver + NaOV |
25.49
|
2.80
|
| |
vanadate-sensitive ATPase Activity (nmol/mg
min)
|
| |
453228 (Old K228) Membranes
|
453200 (Old K200) Membranes
|
| Buffer |
7
|
4
|
| Verapamil |
32
|
3
|
2. ATPase Assay: Drug Concentration
- Dependence
This assay is a modification of the methods of Sarkadi et al., (1992)
J. Biol. Chem. 267:4854 and Druekes et al., (1995) Analyt. Biochem. 230:173.
Assay each drug with orthovanadate, and without orthovanadate, at 0 and
20 minutes each ( = 4 conditions), in duplicate or triplicate wells/condition.
The drug concentration range is dependent on the specific drug. See example
below.
|
Human Pgp-ATPase Assay
Example
96-well Plate Set-Up:
Drug Concentration – Dependence
(Concentration in assay)
|
| |
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
|
11
|
12
|
|
| |
Phosphate
Standards
(nmoles/well)
|
T
|
E
|
S
|
T
|
|
D
|
R
|
U
|
G
|
+ Control
|
|
| |
|
|
0uM
|
2uM
|
10uM
|
20uM
|
40uM
|
60uM
|
80uM
|
120uM
|
150uM
|
Verap
|
|
|
A
|
0
|
0
|
Membranes without sodium orthovanadate
|
t=0 |
|
B
|
3
|
3
|
t=0 |
|
C
|
9
|
9
|
t=20 |
|
D
|
30
|
30
|
t=20 |
|
E
|
60
|
60
|
Membranes with sodium orthovanadate
|
t=0 |
|
F
|
90
|
90
|
t=0 |
|
G
|
120
|
120
|
t=20 |
|
H
|
150
|
150
|
t=20 |
Assay Method
| 1. |
Prepare Phosphate Standards:
These are used to determine the amount of inorganic phosphate liberated
from the hydrolysis of ATP. Prepare 10mM Potassium Phosphate by diluting
the 0.1M Potassium Phosphate, pH 7.4 in Tris-Mes (1:10). Use this
working stock to make: 0.05, 0.15, 0.50, 1.0, 1.5, 2.0, and 2.5mM
phosphate in Tris-Mes. These solutions will be added in 60ul aliquots
to the plate, (along with Tris-Mes buffer alone) for standard solutions
of: 0, 3, 9, 30, 60, 90, 120, and 150 nmoles of phosphate. Add 60ul
in duplicate wells to the assay plate. |
| 2. |
Prepare drug(s) to be tested in Tris-Mes
buffer and load 20ul, in duplicate or triplicate to
the plate. Prepare solutions of the drug to be tested at three times
the desired final concentration. (Twenty-ul aliquots are added to
the plate and then diluted 1:3 upon the addition of 20ul of diluted
Pgp membranes and 20ul of MgATP).
One application of the ATPase assay, is to determine a drug concentration
range of interaction with Pgp. Each dilution of the drug must be tested
with and without the presence of 100uM sodium orthovanadate*, at both
0 and 20 minute incubation with Pgp and MgATP, in duplicate or triplicate
wells (i.e. 8 or 12 wells are needed per drug concentration). One
method, is to prepare: one solution of drug, one solution of membranes
with orthovanadate, one membrane solution without orthovanadate, and
the MgATP as another.
*Orthovanadate inhibits Pgp by trapping MgADP in the nucleotide binding
site. |
| 3. |
Prepare 60 uM Verapamil (positive control for the assay)
in Tris-Mes Buffer,. and load 20ul aliquots (final concentration in
the assay is 20uM) in duplicate or triplicate to the plate |
| 4. |
Remove the Pgp membranes from -80° C,
and prepare the membrane solution(s) at 2mg protein/ml in Tris-Mes
buffer, with or without sodium orthovanadate at 300uM. Load 20ul of
the membrane solutions to all wells except those containing the phosphate
standards. |
| 5. |
Incubate the plate containing the phosphate
standards, buffer +/- drugs, and membranes at 37°C, for 3 to 5
minutes. |
| 6. |
Prepare MgATP in Tris-Mes at 3X the desired
final concentration, while plate is incubating. The final concentration
of MgATP in in the wells should be 3-5mM, so prepare MgATP at 12-15mM.
Load 20ul of the MgATP solution to the t=20min wells. (Do not add
MgATP to the wells which contain the phosphate standards). Use 20
second intervals between row or column additions of MgATP. The stop
solution (10% SDS) will be added in the same order with the same intervals
later. Place the remaining MgATP solution on ice. This will be added
to the t=0 wells later. |
| 7. |
Incubate the plate at 37°C, for 20 minutes. |
| 8. |
During the incubation, prepare 10% Ascorbic
Acid in dH2O at pH 5.0. This solution will be used with the Ammonium
Molybdate solution to prepare the detection reagent. For one assay
plate, a minimum of 20 mls of ascorbic acid is needed. Prepare this
solution freshly for each assay. |
| 9. |
Prepare a reagent reservoir filled with the stop solution
(10% SDS containing Antifoam A), and set and prepare the multichannel
pipettor for the addition of 30 ul of stop solution. |
| 10. |
Remove the plate from 37°C at 20 min (t=20), and
add 30ul of stop solution to the t=20 wells. Use the same addition
order and intervals as was used in adding the MgATP (step 6). |
| 11. |
Add 30ul of stop solution to the t=0 wells, and the
standards/blk wells, so that every well in the plate has received
the solution. |
| 12. |
Add 20 ul of the MgATP solution to the t=0 wells. |
| 13. |
Prepare the detection reagent: 1 part 35mM Ammonium
Molybdate and 4 parts fresh 10% Ascorbic Acid at pH 5.0 (e.g. 5ml
Ammonium Molybdate into 20ml Ascorbic Acid). |
| 14. |
Add 200ul of the detection reagent to all wells in the
plate with a multichannel pipettor. |
| 15. |
Incubate plate for 20 min at 37°C, and
read absorbance between 630 and 850 nm on a microplate spectrophotometer.
|
3. Analysis of ATPase
Assay Data
Calculations
| 1.
|
Construct a phosphate standard curve (phosphate standards (nmoles)
vs. absorbance value) using linear regression analysis. |
| 2. |
Calculate
the nmoles of phosphate released in the samples, from the phosphate
standard curve. |
| 3. |
Within
a set of conditions, subtract the nmoles of phosphate released in
the t=0 samples from the t=20 samples to yield the nmoles of phosphate
released in 20min, in the samples with vanadate and the samples without
vanadate. |
| 4. |
Subtract
the nmoles of phosphate released in 20 min in the samples with vanadate
from the samples without vanadate to give vanadate-sensitive release
of phosphate in 20 min. |
| 5. |
To calculate
the vanadate-sensitive ATPase Activity (nmol/mg min), divide the vanadate-
sensitive release of phosphate in 20 min by the number of mgs of protein
present in each well and the time of the incubation. See example below.
|
| Example: 0.02 ml |
x 2 mg/ml Pgp |
|
| |
membrane = |
0.04mg protein |
| |
(0.04) (20) = |
0.8 mg min |
|
