Cells were centrifuged at 400 g for 1 min at 4°C, washed twice with 1 ml of Wash buffer and then re-suspended in 800 μl of Breakage buffer (5 mM Tris HCl pH 7.4, 20 mM KCl, 2 mM EDTA-KOH pH 7.4, 0.4 M sorbitol, 1% thiodiglycol, 125 mM spermidine, 50 mM spermine).
Resin was washed four times with 60 mL of wash buffer (20 mmol/L Tris-HCl, 500 mmol/L NaCl, 25 mmol/L imidazole, pH 8.5) and eluted with 30 mL of wash buffer supplemented with 1 mol/L imidazole.
Beads were washed four times with 1 ml of wash buffer (200 mM Tris at pH 8.0, 100 mM NaCl and 0.5% NP-40), once with ice-cold phosphate buffered saline (PBS), and boiled in 2× loading buffer.
The pellet was washed with 1 ml of wash buffer (Dulbecco's PBS, Invitrogen).
Plant incubated with 20 ml of wash buffer were used as a control.
After immunoprecipitation, the beads were washed 4 times with 1 mL of wash buffer.
Next, membrane was washed with 15 ml of wash buffer (PBS, 0.05% Tween 20) twice for five minutes each.
After 30 min the cells were centrifuged at at 400 x g and resuspended in 1 ml of wash buffer.
Samples were again centrifuged at 10,000 rpm for 15 sec, flow throughs discarded, and 0.350 mL of wash buffer (RWI buffer; Qiagen) added to each mini-column.
The membrane was again washed with 15 ml of wash buffer (PBS, 0.05% Tween 20) three times for five minutes each.
The next day the membrane was washed with 15 ml of wash buffer (PBS, 0.05% Tween 20) three times for five minutes each.
