Biomedical Engineering Reference
In-Depth Information
Then dilute them 10 times in cell culture medium to obtain
the concentrations: 1 mM, 100
μ
M, 10
μ
M, 1
μ
Mand
μ
μ
0
M. Add 2
l of these solutions to the wells containing
200
l of cell suspension. The following final concentrations
will be obtained: 10
μ
M,100and10nMand0nM
(
see
Note 13
). After treatment, cells must be incubated for
at least 30 min with the inhibitor at 37
◦
C.
6. Centrifuge the plate at 200
μ
M, 1
μ
g
for 5 min at 4
◦
C. Discard the
supernatant by aspiration and add 20
×
l of lysis buffer. Mix
thoroughly and leave the plate on ice for 30 min for the lysis
to take place. Centrifuge the plate at 2500
μ
g
for 15 min at
4
◦
C and transfer the lysate to a fresh plate leaving behind the
pellet.
7. At this point the protein content of the cell lysates should be
measured using standard proteins quantification procedures.
8. Keep the cell lysates on ice until performing the Aktide assay
(
see
Note 14
).
×
1. Prepare the reaction mix by adding 200
μ
l of reaction buffer
3.2.AktideAssay
to 150
μ
l of Aktide stock + 650
μ
l water in a test tube.
2. Distribute 5
l of the reaction mix in the wells of a 96-
well plate (V-shaped bottom) and start the assay by adding
2.5
μ
μ
lofcelllysate(
see
Note 15
). Mix the contents thor-
oughly by pipetting up and down five times. The final con-
centrations of each substrate are 100
μ
M ATP and 100
μ
M
Aktide.
3. Incubate the reaction for 10 min at 37
◦
C shaking gently.
Stop the reaction by adding 100
μ
lof0.05
μ
M IS peptide
dissolved in stop solution (
see
Note 16
).
3.3.Product
ExtractionbySCXin
96-WellPlateFormat
1. Use 2.5
μ
l of Dynabeads
R
SCX (50% slurry) per reaction,
l(
see
Note 17
).
2. Conditioning and washing the beads can be done in bulk.
For this purpose transfer the beads to a 1.5 ml protein low-
bind Eppendorf tube. The magnetic beads are separated
from the solvent using a magnetic rack for Eppendorf for
1 min, discarding the solvent afterwards (this applies to all
step when separation is required).
3. Add 800
for 100 reactions use 250
μ
l of conditioning solution (1 M NaCl, 50 mM
ammonium bicarbonate, freshly prepared) to the beads and
incubate them for 10 min at room temperature (RT) (
see
Note 18
).
4. Wash the beads three times in 800
μ
μ
l of loading solution
(25% ACN, 0.1% TFA, 0.01% Tween-20) (
see
Note 19
).
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