Biomedical Engineering Reference
In-Depth Information
2.2 Kinase Assay
1. Kinase buffer: 25 mM Tris-HCl pH (7.4), 5 mM
-glycero-
phosphate, 10 mM magnesium chloride, 2 mM DTT, 0.1 mM
sodium orthovanadate, 200
ʲ
ʼ
M ATP, 12.5
ʼ
g/ml bovine
serum albumin (
see
Note 2
).
2. Recombinant Akt (
see
Note 3
).
1. Tris-buffered saline with Tween-20 (TBST): 1 l 1× TBS, 1 ml
Tween-20 (
see
Note 4
).
2. Blocking solution: 5 % (w/v) bovine serum albumin in TBST
(
see
Note 5
).
3. Antibody diluting solution: 5 % (w/v) bovine serum albumin
(BSA) in TBST.
4. Primary antibodies: Anti-FLAG and Anti-phospho-Akt sub-
strates (Cell Signaling Technology) (
see
Note 6
).
5. Secondary antibody: Anti-rabbit and anti-mouse conjugated
to horse radish peroxidase (HRP).
6. Immobilon™ Western Chemiluminescent HRP Substrate detec-
tion system (ECL, Millipore) and LAS 500 Western imaging
system (
see
Note 7
).
2.3 Western Blotting
2.4 Identifi cation
of Known
Phosphorylation Sites
and Prediction
of Potential Kinases
1. PhosphoSitePlus (phosphosite.org).
2. Scansite (scansite.mit.edu).
3
Methods
To directly determine that Akt phosphorylates Sec24, epitope-
tagged Sec24 is immunoprecipitated and a kinase assay with recom-
binant Akt is performed. The samples are then subjected to Western
blotting with an antibody specifi c to phosphorylated substrates of
Akt.
See
Fig.
2
for an overview of this process.
To identify known phosphorylation sites in a protein of inter-
est (in our example, DHCR24), a database, PhosphoSite [
16
], is
examined. To determine potential kinases that may phosphorylate
DHCR24, a prediction program, Scansite [scansite.mit.edu, [
17
]],
can be employed.
1. Culture Chinese Hamster Ovary-7 (CHO-7) cells in a 10 cm
dish in DMEM/F12 supplemented with 5 % lipoprotein defi -
cient serum prepared from newborn calf serum (
see
Note 8
).
2. After 24 h, transfect cells with a plasmid encoding Sec24C
fused to a FLAG epitope tag.
3. After another 24 h, rinse cells twice with ice cold PBS.
3.1 Immunopreci-
pitation (IP) of Protein
