A. For MDCK cells use cells in one near confluent well of a 6 well dish.
B. For Rat2 cells use cells from one near confluent 100mm dish.
Remove the medium.
Add 1ml PBS and scrape off the cells, transfer to a microfuge tube.
Spin down gently: if you have an adjustable speed microfuge set the speed between 1 and 2 and spin for 5mins, in a non-adjustable speed
microfuge pulse for 2 x 2secs.
Remove as much liquid as possible then resuspend the cell pellet in 25æl cold NP40 lysis buffer, place on ice for 5min.
Spin down harder: full speed for 10secs.
Remove supernatent to a fresh tube containing 25æl 2 X Tricine gel loading Buffer (TLB).
Either run directly on a Tricine gel and western blot for your expressed protein or store at
-20ºC until you have enough samples ready to run gels.
NP40 Lysis Buffer
50mM Tris pH 8.0
150mM NaCl
1% NP40
4X protease inhibitors (PIN)
2 X TLB: see under Tricine gels p14.
200 X PIN: see under Transcription/Translation p18.