ULTIMATE FREEZE-THAW LYSIS FOR MAMMALIAN CELLS

This extraction procedure was developed by the Simmlaboratory and described in Rudolph et al.,1999(Anal Biochem 269,66-71).It’s a great way to make very concentrated extracts for Western blotting,and is particularly useful when studying low-abundance proteins (like Myc!).The protocol is described for harvesting 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 (45s in cold-room).

4.Resuspend cell pellet in FT LYSIS Buffer (25-40 μl per confluent 6 cm dish,50-80 μl per confluent 10 cm dish).Resuspend by pipeting up and down.

5.Drop tube in liquid nitrogen to freeze.Thaw sample on ice (thawing takes a few minutes).Vortex briefly.

6.Repeat freeze-thaw cycle two more times.

7.Thaw cell lysate.Add 250 U of Benzonase (Merck)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 95℃ for 10 minutes.Load immediately on gel.(Note that these samples must be loaded while still warm,because the salt in the FT Buffer will cause the SDS in the Laemmli to precipitate when cold).

B.SOLUTIONS.

FT LYSIS BUFFER For 100 ml

600 mM KCl20 ml 3 M KCl

20 mM Tris-Cl (pH 7.8)2 ml 1 M Tris-Cl (pH 7.8)

20% Glycerol 20 ml glycerol

Add fresh each time:

0.4 mg/ml Pefabloc 1:100 of 40 mg/ml stock

10 μg/ml Leupeptin 1:1000 of 10 mg/ml stock

10 μg/ml Pepstatin 1:500 of 2 mg/ml stock

5 μg/ml Aprotinin 1:2000 of 10 mg/ml stock