Biomedical Engineering Reference
In-Depth Information
protocol allows cAMP-dependent phosporylation as well as other
enzymatic protein phosphorylations.
A
480 mM KCl, 160 mM histidine, 40 mM MgCl
2
,pH6.8
Solutions/Reagents
B .5mM Tris-ATP
2,3
C[
γ
/µ
-
32
P]ATP in Soln. B 10 - 20 kBq
l
D .5M NaF in ddH
2
O
E2mM EGTA
F 0
µ
/
g
ml catalytic subunit of cAMP PrK
µ
/
ml cAMP PrK inhibitor
4
G 0
g
µ
/
H 0
g
ml calmodulin
I
mM CaCl
2
J
mM KCl, 20 mM Tr i s
·
HCl, 0.2 mM DTEorDTT,pH6.8
K
15% trichloroacetic acid (w/v), 50 mM sodium phsophate
in water
Pipet the assay according to Table 6.7, cool the tubes in an ice
bath, then add the protein solution or membrane suspension which
Table 6.7.
Protocol for phosphorylation with protein kinases (PrK)
Type of protein kinase
Solution
ADE F GH I
(
µ
µ
l per 200
l assay)
Exogenous cAMP PrK
50
10
10
10
-
-
-
Control
50
10
10
-
10
-
-
10
a
Endogenous calmodulin-
50
10
-
-
10
10
sensitive PrK
Control
10
a
50
10
10
-
-
-
Endogenous PrK in the
50
10
-
-
10
-
10
presence of endogenous
calmodulin
Control
50
10
10
-
10
-
-
Other endogenous PrK
b
50
10
10
-
-
-
-
Control
50
10
10
-
10
-
-
a
Omit the inhibitor of cAMP PrK if this enzyme is not present or if its
activity is part of the control
b
Potential activators of endogenous protein kinases, e.g. cycloAMP, cy-
cloGMP, inositol phosphates, or lipids may be added in physiological
concentrati
ons
2
PourasolutionofsodiumATPontoasmallcolumnofacationexchange
resin, equilibrated with Tris. Elute Tris-ATP with ddH
2
O and monitor
by UV absorption at 260 nm.
3
-
32
P]ATPisnotveryhighorif
endogenous phosphatases are absent, the final concentration of 250
γ
If the specific radioactivity of the [
µ
M
AT P m ay b e r e d u c e d .
4
Preparation of cAMP PrK inhibitor according to Demaille JJ, Peters KA,
Fischer EH (1977) Biochem 16:3080
