核心提示：This extraction procedure was developed by the Simm Laboratory and described in Rudolph et al., 1999. It’s a great way to make very concentrated extracts for Western blotting, andis particularly useful when studying low-abundance proteins . The protocol is described forharvesting mammalian cells from 6 or 10 cm tissue-culture dishes.
A. CELL HARVEST.1. Remove media from cells. Wash in ice-cold PBS.2. Scrape cells in 1 ml of ice-cold PBS. Transfer to 1.5 ml microfuge tube.3. Pellet cells at top speed in microfuge .4. Resuspend cell pellet in FT LYSIS Buffer . Resuspend by pipeting up anddown.5. Drop tube in liquid nitrogen to freeze. Thaw sample on ice . Vortex briefly.6. Repeat freeze-thaw cycle two more times.7. Thaw cell lysate. Add 250 U of Benzonase to digest DNA.Mix. Incubate at room temperature for 10 minutes.8. Measure protein concentration.9. Add an equal volume of 2xLaemmli Buffer prior to analysis by SDSPAGE.Heat at 95oC for 10 minutes. Load immediately on gel. .
B. SOLUTIONS.FT LYSIS BUFFER For 100 ml600 mM KCl 20 ml 3 M KCl20 mM Tris-Cl 2 ml 1 M Tris-Cl 20% Glycerol 20 ml glycerolAdd fresh each time:0.4 mg/ml Pefabloc 1:100 of 40 mg/ml stock10 μg/ml Leupeptin 1:1000 of 10 mg/ml stock10 μg/ml Pepstatin 1:500 of 2 mg/ml stock5 μg/ml Aprotinin 1:2000 of 10 mg/ml stock