小中大
A general protocol for sample preparation is described below.
Treat cells by adding fresh media containing regulator for desired time.
Aspirate media from cultures; wash cells with 1X PBS; aspirate.
Lyse cells by adding 1X SDS sample buffer (100 μl per well of 6-well plate or 500 μl per plate of 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
Sonicate for 10–15 seconds for complete cell lysis and to shear DNA (to reduce sample viscosity).
Heat a 20 μl sample to 95–100°C for 5 minutes; cool on ice.
Microcentrifuge for 5 minutes.
Load 20 μl onto SDS-PAGE gel (10 cm x 10 cm). NOTE: CST recommends loading prestained molecular weight markers (#7720, 10 μl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 μl/lane) to determine molecular weights.
Electrotransfer to nitrocellulose or PVDF membrane.
这是在CST公司网页上的protocol其中一部分，其中裂解细胞就用1X SDS sample buffer ，我应该没看错吧，这是蛋白loading buffer吧，难道没有人知道吗，但是没有测浓度，不知道怎么蛋白定量，不知道有没人这样做啊，第4步可以省去吗
