Biology Reference
In-Depth Information
B-like protein family. Some calcineurin B-like family proteins are
reported to regulate transporter functions by connecting calcineu-
rin B-like protein-interacting protein kinases with target trans-
porter proteins [
9
].
1. Prepare AtCBL1 mRNA by in vitro transcription as described
above (Subheading
3.1
).
2. Thaw WG extract (WEPRO 1240), creatine kinase, and dialy-
sis buffer on ice. Keep all reagents on ice while in use. Freeze
promptly afterwards. Prepare 25
μ
l of reaction mixture on ice
as follows: 6.25
μ
l of WG extract, 0.25
μ
l of 40 mg/ml cre-
atine kinase, 4.7
μ
l of 4× dialysis buffer, 0.5
μ
l of [
14
C]myristic
l of Milli-Q water.
3. Incubate the reaction mixture at 26 °C for 3 h.
acid, 7.5
μ
l of mRNA, and 5.8
μ
3.12 Detection of
Myristoylated AtCBL1
with Imaging Analyzer
1. Subject the reaction mixture to SDS-PAGE.
2. After electrophoresis, dry the gel with a gel dryer.
3. Put the dried gel into the BAS cassette2 2040 with the imag-
ing plate.
4. Visualize the labeled proteins by the imaging analyzer (Fig.
4a
).
3.13 Bilayer
Synthesis of
Myristoylated AtCBL1
in the Presence of
[
14
C]Leucine
1. Prepare AtCBL1 mRNA by in vitro transcription as described
above (Subheading
3.1
).
2. Thaw WG extract (WEPRO 1240), creatine kinase, and dialy-
sis buffer on ice. Place and keep all reagents on ice. Prepare
25
μ
l of translation mixture on ice as follows: 6.25
μ
l of WG
extract, 0.25
μ
l of 40 mg/ml creatine kinase, 4.7
μ
l of 4× dialy-
sis buffer, 0.5
μ
l of 3.75 mM myristic acid, 0.5
μ
l of [
14
C]leu-
cine, 5
μ
l of 50 mg/ml liposomes, 7.5
μ
l of mRNA, and 0.3
μ
l
of Milli-Q water.
3. Prepare 125
μ
l of substrate mixture on ice as follows: 31.25
μ
l
of 4× dialysis buffer, 2.5
μ
l of 3.75 mM myristic acid, 2.5
μ
l of
[
14
C]leucine, and 88.75
μ
l of Milli-Q water.
4. Transfer 125
l of substrate mixture to a microtiter plate.
5. Carefully pipette the 25
μ
l of translation mixture underneath
the substrate mixture to form the bilayer reaction.
6. Seal the plate to avoid evaporation.
7. Incubate the plate at 15 °C for 24 h.
μ
Accudenz is a nontoxic regent used for fractionation of proteins,
organelles, and cells [
10
-
12
]. Accudenz density ultracentrifuga-
tion separates liposomes from WG extract proteins. The liposomes
in the CF reaction mixture fl oat to the top of the tube as their
contained buffer is less dense than Accudenz.
N
-myristoylation of
AtCBL1 is confi rmed by co-fl oatation of protein with liposomes.
3.14 Accudenz
Density Gradient
Ultracentrifugation
