Protein expression was induced in 96-well deep well block, and cell pellets were frozen.

• Thaw cells
• Resuspend cells in 100 µl of
• 50 mM Tris-HCl, pH 8.0
• 0.3 M NaCl
• 0.1 mM EDTA
• add 25 µl of
• 5 µl lysozyme 10 mg/ml
• 12.5 µl 1% Brij 58
• 7.5 µl resuspension buffer

µl per well
5	lysozyme 10 mg/ml
12.5	1% Brij 58
7.5	resuspension buffer

• Incubate on ice 30 min
• Add 25 µl of
• 0.3 µl MgCl₂, 1 M
• 0.1 µl Benzonase gradeII 25 U/µl Merck
• 24.6 µl 50 mM Tris-HCl pH 8.0

µl per well
0.3	MgCl2, 1 M
0.1	Benzonase 25 U/µl
24.6	50 mM Tris-HCl pH 8.0

• vortex briefly, incubate at room temperature 30 min
• centrifuge lysates 30 min 6200 rpm 4°C
• transfer supernatants to filter plate
• fiter supernatants

• add 15 µl 0.1 M imidazole (endconc. 10 mM)
• add 25 µl of 20% NiNTA agarose equilibrated in 50 mM Tris, pH 8.0
• shake 30 min room temperature (better would be: 1 hour cold room)
• remove liquid
• wash three times with
• 50 mM Tris-HCl, pH 8.0
• 0.3 M NaCl
• 20 mM imidazole
• elute with 25 µl of
• wash buffer containing 250 mM imidazole