Sf21 extraction
1. Cells are harvested (p60 dishes) at approx. 48 hrs post infection. Cells are most easily
collected in their medium using a pasteur pipet to gently blow the cells off the dish.
Centrifuge 1200 rpm 5 min. Aspirate medium carefully, not disturbing the pellet.
2. Extract the cell pellet: Use 500 µl TEB supplemented with protease inhibitors (PMSF,
leupeptin, pepstatin) and phosphatase inhibitors (1 mM NaF, 10 mM -glycerophosphate,
0.5 mM Na3VO4) for 15 min on ice. Centrifuge 10 min in microfuge at 10,000xg.
Transfer cell lysate to a new tube.
1. Sf21 infected with HA-mBub1: IP neg contrl with 2 µl 9E10
2. Sf21 infected with HA-mBub1: IP pos contrl with 2 µl HA.11
3. Sf21 infected with GST-Bub1 600: Bring down with GSH beads
4. Sf21 infected with GST-Bub1 600: Bring down with GSH beads
Do IP’s or GSH-bead pull-downs and wash so that you have purified HA-Bub1 or GSTBub1 600 linked to beads.
3. In vitro kinase reaction. Make up kinase buffer: 50 mM Tris pH 7.5, 10 mM MgCl2, 1
mM NaF, 10 mM -glycerophosphate, 1 mM DTT. Wash IP’s or pull-downs twice with
kinase buffer. Add 40 µl of His-p53 (dialyzed) to the beads and supplement until 1 mM
NaF and 10 mM -glycerophosphate. Add 100 mM ATP (cold) until 100 µM final
concentration. The final volume should be about 50 µl or less. Allow in vitro kinase
reaction to proceed at 30 ˚C for 30 min. Do also a kinase reaction with only the His-p53,
no beads added (neg control reaction).
4. When the kinase reaction is finished, spin down and transfer the supernatant containing
the phosphorylated p53 product into a new tube. Add 25 µl 2xDB with reducing agent.
Boil 5 min and load the whole volume in one lane of a long SDS-PAGE gel. Run
overnight.