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Table 2 Summary of the purification procedure

From: Use of anionic denaturing detergents to purify insoluble proteins after overexpression

Step Description Purpose Comments
1 Growth, induction and harvest of induced cells Overexpression of target protein in E. coli As described elsewhere [23]
2 Resuspension in 1% SDS in PBS Lysis of cells Works fine in 1/20 to 1/10 of the original culture volume. Other buffers also work (50 mM Tris/Cl pH 7.0, 100 mM NaCl and 1% SDS)
3 Sonication Solubilisation of proteins from inclusion bodies Until solution turns clear. Often faster than 2 minutes.
4 Incubation on ice for 30 minutes Precipitation of SDS Precipitation of SDS apparent after 5 minutes. 1 h incubations on ice possible.
5 Centrifugation Removal of precipitated SDS SS34 rotor, 13 krpm, 20 minutes, 4°C
6 Ni/NTA affinity purification Capture and washing of hexahistidine tagged target protein See M&M for buffer compositions and details.
7 Elution in 0.1% Sarkosyl Elution in a dialyzable detergent that is compatible with refolding [24] Other detergents could be used but were not tested.
  1. Key steps in the procedure are described and their purpose briefly explained. The comments are based on the experiments described herein and additional purifications using the same principle but with slightly different protocols (altered buffers, volumes and incubation time on ice, data not shown).