Biomedical Engineering Reference
In-Depth Information
and absence of the drug, “P
i
” and “P
i
”, blank B, and amount of
used Protein “a” (mg) according to the equation
µ
molP
i
µ
molP
i
−
µ
=
kat Na,K − ATPase/mg
900
Determination of inorganic phosphate
(see Protocol 1.3.2)
Mix 0.50 ml aliquots of the ATPase assay and phosphate standards
(from 10 mM KH
2
PO
4
(
0.5
·
a
·
=
/µ
l) in the range between 50 and
350 nmoles P
i
per 0.5 ml)with1.50ml Soln. H. Close the reaction
tubes and incubate at 37
◦
C in the dark for 1.5 - 2 h.Readat750nm
and calculate from the standard curve.
10 nMol
5.3.2.2 Receptor Determination: DHP Binding Sites
on Surface Membranes
Surface membranes of excitable cells contain a voltage-dependent
L-type calcium channel, a membrane-spanning complex of several
proteins. This complex is a valuable marker for this membrane
type. It binds very specific calcium channel antagonists, such as,
for instance, drugs of the dihydropyridine (DHP) type as (+)-PN
200 - 110 or nitrendipine.
Important!
DHP's are light sensitive. Avoid daylight and work in
a dark room illuminated by yellow light (e.g., sodium light source).
A 0mM Tr i s , p H 7 . 4 , 2 mM CaCl
2
,0.1mM PMSF (added just
Solutions/Reagents
before use)
=
PN: (+)-[Methyl-
3
H]PN 200 - 110 (100 000 to 120 000 dpm
B
/
µ
1670 to 2000 Bq; specific radioactivity ca. 3 TBq
mmol)in50
l
Soln. A (starting dilution)
C l
µ
: 1
M nitrendipine in Soln. A (final concentration)
D
precipitating agent: 10% PEG 6000 (w/v) in 10 mM Tr i s , p H
7.4, 10 mM MgCl
2
,keptinanicebath
E
0.3% polyethylenimine (PEI) in pure water
Diluteamembranepreparationtoabout0.4mg protein per milliter
or cell suspensions to about 10
6
cells/ml with Soln. A. Prepare four
1:3 dilution series of Soln. B using Soln. A as diluent (dilutions
“0” to “4”). Pipet 50
µ
l of dilutions “0” to “4” as duplicates into
scintillation vials to define the total radioactivity values “Total
0
”to
“Total
4
.”
First pipet Soln. A, B, and C according to Table 5.7 into dis-
posable 4-ml tubes. Then add membrane or cell suspension to
start the ligand binding and incubate at 37
◦
C for 1 h.Putthe
tube into an ice bath and stop by addition of 2 ml each of ice-cold
Soln. D.
Filter the samples on Whatman GF/C glass fiber filters, wetted
with Soln. E, in a holder manifold and wash twice with 2 ml of
ice-cold Soln. D. Transfer the filters into scintillation vials, add
scintillation cocktail, and count for radioactivity.
