South-Western

Southwestern-Blot (Klempnauer and Sippel,1987, EMBO J. 9, 2719-2725)

- run SDS-PAGE, transfer protein to nitrocellulose

- check transfer by staining wih Ponceau S (optional)

- destain by rinsing blot with PBS, then

- incubate three times in 6 M urea, 0.2% NP-40 for 20 min at room temperature on shaker

- incubate in DNA binding buffer:

10 mM Tris/HCl pH 7.8
1 mM EDTA
50 mM NaCl
four times for 30 min at room temperature on shaker (blot can be stored at 4˚C overnight in DNA binding buffer)

- incubate blot in DNA binding buffer with radioactively labelled DNA (40 ng-80 ng/ml for endlabelled DNA, 10 ng - 20 ng/ml for nick-translated DNA) 30 min at room temperature with shaking

- wash three times with DNA binding buffer (- DNA) for ten minutes at room temperature, briefly dry blot, expose.