Prepare mutant and wild-type DNA using Qiagen maxi-prep. Measure ODs very carefully so that equal amounts can be applied to cells.
For HeLa cells the most effective transfection method is CaPO
4, efficiency >30% is standard and can be checkedwith a b-galactosidase construct.Transfect cells at ~70% confluency, it is important to have well distributed cells and not large clumps (this can seriously impair transfection efficiency). If the cells don't look good- abandon ship- set up new cells.
Remove CaPO
4 after 16-18 hours, let cells recover in complete medium for 4 hours and then change medium to 0.5% serum and leave for 24 hours (alternatively change to 0% serum and leave for 4-6 hours).Make up EGF in DMEM (no serum)(100ng/ml), 5ml per 10 cm dish.
pre-warm this solution (37°C)
Take off medium from cells and add EGF solution, incubate in the incubator for 8 minutes.
rapidly rinse twice with cold PBS, (dishes on a cold plate).
Add cold lysis buffer (1ml per 10 cm dish), rock gently on a cold plate for 2 minutes.
Lysis buffer
0.5% (w/v) NP40
25mM Tris-HCl, pH 7.5
100mM NaCl
0.1mM EDTA
50mM NaF
1mM Na
3VO4 (check protocol of how to prepare)1mM PMSF
protease inhibitor cocktail
Collect lysate into labelled cooled eppendorfs, spin for 10 minutes at 10,000rpm in a refrigerated microfuge.
collect and save a 50µl sample of the supernatant into pre-labelled tubes.
take rest of the supernatant into fresh eppendorfs (pre-label) containing 5µl PY20 antibody.
Rotate end over end for 1 hour, 4°C and then add 40µl of protein A sepharose for a further hour.
Spin so that beads pellet , collect and retain supernatant.
wash pellet x4 with wash buffer. Remove most of supernatant. Spin. Take off last vestiges of final supernatant very carefully using a gel loading tip.
Wash buffer
0.1% NP40
10mM Tris-HCl (pH 7.4)
150mM NaCl
1mM Na
3VO4resuspend pellet in 60µl of SDS-PAGE buffer- heat and spin. Apply 20-40µl to gel.