Protocols

Ni NTA Purification

Pellet

• centrifugation of the bacterial culture for 10 min at 8000 rpm at 4 °C
• discard the supernatant

Lysis

• per 1g wet weight of the pellet add 4ml lysis buffer
• fill up to 0.5 mg/ml with lysozyme
• incubation on ice for 2 h
• centrifuge for 30 min at 10000 g at 4 °C

Column

• equilibrate the column with 0.5 ml Ni-NTA (0.25 ml bed volume)
• add 0.5 ml lysis buffer and slightly centrifuge the buffer through the column, it has to stay wet (do twice)
• pump the cell lysate (supernatant) for 1 h at ice through the column

Wash

• twice with 1.25ml wash buffer
• four times with 0.5 ml elution buffer
• revover the supernatant

Load a sample of every step in a SDS gel.
All buffers come from the Qiagen "Ni-NTA Agarose Purification of 6xHis-tagged Proteins from E. coli under Native Conditions - Quick-Start"-protocol.