Biomedical Engineering Reference
In-Depth Information
filter filters (
→
“B
t
”). Count for radioactivity and plot B
t
/B
0
vs
time t.
Dilute 2
10
7
cellsin17ml Soln. A and warm up to 37
◦
C.
On-kinetics
Prepare a solution of
3
H-PN 200 - 110 (about 1 600 000 dpm)in
3 ml. Mix both solutions and continue incubation at 37
◦
C.Take1-
ml probes in duplicate at time t
×
=
0, 1, 2, 4, 6, 8, 10, 12, 15, and 20 min
after mixing (
“B
t
”). Precipitate the probes with 2 ml of ice-cold
Soln. D immediately after sampling and filter on Whatman GF/C
glass fiber filters. Prepare a blank (“B
0
”) by incubation of a mixture
of 850-
→
µ
µ
l Soln. C at 37
◦
C for 20 min.
Wash the filters with Soln. D. Dry the filters at air and count for
radioactivity.
Plot B
t
/B
0
vs t and calculate the maximal number of binding
sites B
max
and the dissociation constant K
D
using either a computer
program(e.g.,Prism)oraScatchard plot (see Chap. 9.2).
If B/F (B, bound ligand PN: B
l cell suspension and 150
=
B
t
−B
0
;F,freeligandPN:
=
F
total PN − B) is plotted vs F, a straight line of the function
B
F
=
a+b
·
B
=
/
=
is obtained. The x-axis intercept (y
B
F
0) represents B
max
;the
slope b gives K
D
.
The association rate constant k
1
is calculated from the slope of
the function B/B
0
=
f(t), the dissociation rate constant k
−1
is the
slope of the equation
/
=
ln(B
B
0
)
−k
−1
·
t
(for details see Chap. 9.2.1)
References
Glossmann H, Striessnig J (1990) Rev Physiol Biochem Pharmacol 114:1
5.3.3 RNA Separation by Non-Denaturating Sucrose Density
Gradient Centrifugation
A1M NaCl, 0.1 M Tr i s
·
HCl, 5 mM EDTA, pH 7.5
Solutions/Reagents
15% sucrose (RNase free) (w/v) in Soln. A
3
B
C
30% sucrose (RNase free) (w/v) in Soln. A
Prepare a linear gradient from Soln. B and Soln. C in a centrifuge
tube (e.g., 13 ml Ultra-Clear for Beckman SW 41 rotor; 6 ml of
Soln. B and Soln. C each) at the day before run. If a gradient mixer
is used, e.g., according to Fig. 2.2, place the centrifuge tube either
in a slanting position, start with Soln. C, and let flow along the tube
3
Boil Soln. B and C in the presence 0.5% diethylpyrocarbonate (v/v) on
the day before use.
