266
Transfer 200 μl of supernatant into a new Eppendorf tube and
add 50 μl 5× SDS-loading buffer (see Note 16), next boil the
sample for 5 min at 95 °C (see Note 17). The lysates can be
stored at −20 °C when not directly used.
4. For SDS-PAGE, load protein ladder and 10 μl of each sample
onto a 9% SDS-gel with 15 pockets (8.3 × 6.4 cm). Conduct
gel electrophoresis in running buffer at 90 V for approximately 2 h.
5. For the semidry Western blot using a PVDF membrane, the
membrane has to be activated in the first step. Therefore, incubate the membrane 1 min in methanol and then incubate for
another minute in transfer buffer. At the same time, incubate
two Whatman paper per gel in transfer buffer. Break open the
SDS-gel chamber and assemble a “sandwich” consisting out of
Whatman paper -- PVDF membrane -- SDS gel -- Whatman
paper (anode to cathode) in a semidry blotter. Perform Western
blot for 1 h at 0.35 A/SDS-Gel.
6. Afterward block the membrane in TBS-T supplemented with
5% BSA (v/v) for 1 h. Dilute primary antibodies 1:1000 in
TBS-T supplemented with 5% BSA (v/v) and add them to the
membrane. Incubate overnight at 4 °C.
7. Day 3: Wash the membrane thrice with TBS-T for 10 min each
time. Dilute secondary antibody coupled to horse radish peroxidase 1:10,000 in TBS-T supplemented with 5% BSA (v/v)
and incubate membrane for 1 h at room temperature. Again
wash the membrane thrice with TBS-T for 10 min each time.
8. Apply ECL I/ECL II mixture (1:1) onto the membrane incubate for some seconds and detect chemiluminescence with
luminescent image analyzer (Fig. 2).
1. Day 1: Place one of the 24-well plates you transfected/seeded
(see Subheadings 3.1.1 and 3.1.2) the day before into a light
box while the control plate remains in the dark. Afterward irradiate cells with 1.5 μmol/(m
2
s) for 5 min.
2. Switch on safe lights before taking cells out of the incubator to
prevent uncontrolled activation of the optoKinase system.
Subsequently, wash them once with DPBS before adding
200 μl 4% paraformaldehyde cell fixation solution per well
(see Note 18).
3. Let the cells incubate for 10 min on ice and subsequently for
additional 10 min at room temperature. Afterward, the samples do not have to be handled under safe light anymore.
4. Apply 7 μl mowiol (with DABCO) onto a microscope slide, try
to avoid generating air bubbles.
5. Use pincers to pick up cover slip and dip it a couple of times
into water to remove excess of paraformaldehyde. Use a cos3.2.2 Fluorescence
Microscopy
Wignand W.D. Mühlhäuser et al.
Transfer 200 μl of supernatant into a new Eppendorf tube and
add 50 μl 5× SDS-loading buffer (see Note 16), next boil the
sample for 5 min at 95 °C (see Note 17). The lysates can be
stored at −20 °C when not directly used.
4. For SDS-PAGE, load protein ladder and 10 μl of each sample
onto a 9% SDS-gel with 15 pockets (8.3 × 6.4 cm). Conduct
gel electrophoresis in running buffer at 90 V for approximately 2 h.
5. For the semidry Western blot using a PVDF membrane, the
membrane has to be activated in the first step. Therefore, incubate the membrane 1 min in methanol and then incubate for
another minute in transfer buffer. At the same time, incubate
two Whatman paper per gel in transfer buffer. Break open the
SDS-gel chamber and assemble a “sandwich” consisting out of
Whatman paper -- PVDF membrane -- SDS gel -- Whatman
paper (anode to cathode) in a semidry blotter. Perform Western
blot for 1 h at 0.35 A/SDS-Gel.
6. Afterward block the membrane in TBS-T supplemented with
5% BSA (v/v) for 1 h. Dilute primary antibodies 1:1000 in
TBS-T supplemented with 5% BSA (v/v) and add them to the
membrane. Incubate overnight at 4 °C.
7. Day 3: Wash the membrane thrice with TBS-T for 10 min each
time. Dilute secondary antibody coupled to horse radish peroxidase 1:10,000 in TBS-T supplemented with 5% BSA (v/v)
and incubate membrane for 1 h at room temperature. Again
wash the membrane thrice with TBS-T for 10 min each time.
8. Apply ECL I/ECL II mixture (1:1) onto the membrane incubate for some seconds and detect chemiluminescence with
luminescent image analyzer (Fig. 2).
1. Day 1: Place one of the 24-well plates you transfected/seeded
(see Subheadings 3.1.1 and 3.1.2) the day before into a light
box while the control plate remains in the dark. Afterward irradiate cells with 1.5 μmol/(m
2
s) for 5 min.
2. Switch on safe lights before taking cells out of the incubator to
prevent uncontrolled activation of the optoKinase system.
Subsequently, wash them once with DPBS before adding
200 μl 4% paraformaldehyde cell fixation solution per well
(see Note 18).
3. Let the cells incubate for 10 min on ice and subsequently for
additional 10 min at room temperature. Afterward, the samples do not have to be handled under safe light anymore.
4. Apply 7 μl mowiol (with DABCO) onto a microscope slide, try
to avoid generating air bubbles.
5. Use pincers to pick up cover slip and dip it a couple of times
into water to remove excess of paraformaldehyde. Use a cos3.2.2 Fluorescence
Microscopy
Wignand W.D. Mühlhäuser et al.