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166 Godat et al.
4. The MagneHis Ni Particles are pre-equilibrated and can be added directly to
the sample.
5. Resuspend lyophilized DNase I (cat. no. Z358B, Promega) with 80 μl doubledistilled
water. Mix to dissolve the powder completely. Remove the DNase
solution from the vial and add it to 1 ml of double-distilled water. For each
reaction, use 5.8 μl DNase solution + 64.2 μl FastBreak Cell Lysis Reagent, 10×.
6. In cases of very high expression (e.g., 50 mg/l), up to 2 ml of resin per liter of
culture may be needed.
7. We do not recommend adding 500 mM NaCl to the FastBreak Cell Lysis
Reagent, 10×, as it could result in particle clumping during lysis and binding in
this system.
8. Cells should be removed from the medium before protein purification.
9. The amount of imidazole in the washes can be optimized by titrating from
10–100 mM imidazole. The higher the amount of imidazole used for washing,
the less background. However, some tagged protein may elute off.
10. Resuspend lyophilized DNase I (cat. no. Z358B, Promega) with 80 μl doubledistilled
water. Mix to dissolve the powder completely. Remove the DNase
solution from the vial and add it to 1 ml of double-distilled water. For each
reaction use 8 μl DNase solution + 92 μl FastBreak Cell Lysis Reagent, 10×.
11. If you wish to collect the flow through, place an empty deep-well plate on the
manifold bed. On top of the deep-well plate place the manifold collar and insert
the filtration plate onto the collar before transferring the lysate.
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