Biomedical Engineering Reference
In-Depth Information
Table 5.7.
Protocol for the determination of ligand (PN) binding
Soln. A
Soln. B
Soln. C
Membrane and
cell suspension
µ
(
l)
To t a l
0
-
50
-
-
Blank
0
50
50
50
100
Bound
0
100
50
-
100
To t a l
1
-
50
-
-
Blank
1
50
50
50
100
Bound
1
100
50
-
100
To t a l
2
-
50
-
-
Blank
2
50
50
50
100
Bound
2
100
50
-
100
To t a l
3
-
50
-
-
Blank
3
50
50
50
100
Bound
3
100
50
-
100
To t a l
4
-
50
-
-
Blank
4
50
50
50
100
Bound
4
100
50
-
100
5.3.2.3 Determination of the Dissociation
and Association Kinetics of the DHP Receptors
A more detailed analysis of ligand binding is possible by determi-
nation of the association rate constant (“on-kinetics”) of the dis-
sociation rate constant (“off-kinetics”). The methodology of these
estimations is illustrated for the cardiac DHP receptor.
A 0mM Tr i s , p H 7 . 4 , 2 mM CaCl
2
,0.1mM PMSF (added just
Solutions/Reagents
before use)
=
(+)-[Methyl-
3
H]PN 200- 110 (60 000 to 80 000 dpm
B
1000-
/
1340 Bq; specific radioactivity ca. 3 TBq
mmol) per milliter of
Soln. A
µ
C
M nitrendipine in Soln. B
D precipitating agent: 10% PEG 6000 (w/v) in 10 mMTr i s , p H 7 . 4 ,
10 mM MgCl
2
,keptinanicebath
E .1mM nitrendipine in Soln. A
blank: 6.7
µ
µ
×
10
6
Off-kinetics
For blank value mix 2
cellsin850
l of Soln. A with 150
l
Soln. C and incubate at 37
◦
C for 60 min. Precipitate with Soln. D
and filter on glass fiber filter as described above (t
=
0;
→
“B
0
”).
10
7
cellsin10ml of Soln. B and
incubate at 37
◦
C for 60 min. Add Soln. E to a final concentration
of 1
Simultaneously suspend 2
×
µ
M after this pre-incubation, vortex, and continue incubation.
Take two aliquots of 50
µ
l eachatt
0
(time of nitrendipine addition)
and 1, 2, 5, 7, 10, 15, 20, 25, and 30 min after nitrendipine addition,
precipitate with 2 ml of ice-cold Soln. D and suck off on glass fiber
