Split the cells into 6 well plates 24 h before transfection to get 50-70%
confluence one day after (use 3 ml of 10% FCS DMEM/well). For example, one
10 cm dish has the surface area of one 6-well plate. A confluent 10 cm dish
can be split into 3 6-well plates.
Follow the protocol of the transfection reagent, and transfect the following
for each protein pair:
A-GST + B-Myc
empty-GST + B-Myc
B-GST + A-Myc
empty-GST + A-Myc
Cell lysis
Aspirate medium.
Wash the cells off with PBS.
Pellet the cells, and resuspend in 300 µl of Lysis Buffer.
Keep on ice for 15 min.
Spin down at 13000 rpm for 1 min.
Take supernatant (keep from 30 to 40 microgrammes for straight western).
GST pulldown
To 120 µl of lysate, add 20 µl of glutathione beads (Amersham)
+ 500 µl lysis buffer.
Incubate 1 hour at 4°C.
Wash the beads 3 times with 500 µl of lysis buffer.
Dry the beads with thin tips.
Add 10 µl of Laemli buffer onto the beads.
Boil for 3 minutes before loading the gels.
Gel loading
For each protein pair, run 3 gels as follows:
Gel 1, to be probed with anti-Myc, containing the pulldowns of the 4 transfections.
Gels 2 and 3, to be probed with anti-Myc and anti-GST respectively, containing
straight lysates of the 4 transfections.
Western blotting
Use 0.75% TBST for all steps.
Block for 1 hour at room temp in 5% milk.
Incubate with primary antibody O/N at 4°C.
Wash 3x quick, then 3x 5 minutes.
Incubate with secondary antibody for 30-60 minutes at room temp.