Team:Freiburg/Notebook/modeling

Effector

Effector Control

Targeting

uniBAss

Modeling

Standardization

Material and Methods

65000 cells per well were seeded. 24 h before transfection.
The cells had been transfected at 21:30 h.

To generate time dependent data, one well was treated every 6 h.

t₀= 01:30
t₁= 07:30
t₂= 13:30
t₃= 19:30
t₄= 01:30
t₅= 07:50
t₆= 13:30

Every timepoint the supernant was taken for SEAP measurement and the cells were lysated with RIPA buffer for quantitative western blotting.
The standard western blot procedure was done with 20 µl of the cell lysates.
For quantification of the western blot data, a dot blot was done. A serial dilution of anti-HA (200ng/µl)was prepared.

1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400

After PVDF-membran activation in methanol and a short incubation in tranfer buffer, 2 µl of each dilution have been droped onto the membrane. After that the membrane was indubated at room temerature (1h) for drying.
The detection with the secondary anti-body was done similar to the treatment of the dCas9 blots.

Results

Figure 1: Western Blot The dCas9 fusion protein possesses a HA-tag and could so be detectet with an anti-HA antibody. The first two blots show dCas9-KRAB. The particular timepoints are labelled above the band. The same pertains for the two dCas9-VP16 blots. As negative control (c), the lysate of timepoint 6 of transfection 3 and 4 has been used.

Figure 1: Dot Blot The dots are labelled with their ditution rate. Only the labelled dots were used for quanitfication. The blots of the first line have been illuminated under the same conditions as the dCas9-KRAB blots, the one of the second line under conditions as the dCas9-VP16 blots.