Add 200ul NP40 Lysis buffer + PIN, place on ice 5mins,
Spin down full speed, 10secs and remove supernatent to fresh tube.
Measure protein concentration.
5A. For MDCK cells run 10ugs on an SDS-Tricine gel, then western blot. Flash freeze the rest and store at
-80ºC.
5B. For Rat2 cells add 5ul anti-mT antibody (mix of 5 monoclonals), incubate on ice 1hour, add
100ul 10% protein G Sepharose in Lysis buffer, place on rotator at +4ºC for 1hour. Spin down 10secs, wash 3 X
500ul Lysis buffer, add 20ul 1XTLB and run on a SDS-Tricine gel. Use the rabbit anti mT serum for the blot.