Isolation Purification and Separation

scientificprotocols authored about 7 years ago

Authors: Chien-Sheng Chen & Heng Zhu 


We have developed a high-throughput protein purification protocol that allows us to purify ~4,000 proteins within 10 hours from prepared culture. By combining steps of cell lysis and protein capture on affinity resins in sealed filter plates, we reduced the number of pipetting steps and thus, human errors.




  1. Re-suspend cell pellets at 4 °C in 80 µL of lysis buffer, containing 50 mM NaH2PO4 at pH 8 with 300 mM NaCl, 20 mM imidazole, CelLytic B (Sigma), Lysozyme (1 mg/mL), Benzonase (50 units/mL), proteinase inhibitor cocktail (Sigma), and PMSF (1 mM/mL).
  2. Wash Ni-NTA Superflow (QIAGEN) twice with water and twice with 50 mM NaH2PO4 at pH 8 with 300 mM NaCl, 20 mM imidazole using centrifugation.
  3. Add 25 μL pre-washed Ni-NTA Superflow (QIAGEN) to each well
  4. Transfer the mixtures into bottom-sealed filter plates (Multiscreen Nylon Mesh)
  5. Seal the filter plates
  6. Incubated for 1.5 hr at 4 °C with vigorously shaking
  7. Remove seals
  8. Wash the resin-protein complexes 3 times with 250 µL/well of wash buffer I (50 mM NaH2PO4 with 300 mM NaCl, 10% glycerol, 20 mM imidazole, 0.01% Triton X-100, at pH 8) and 3 times with Wash buffer II (50 mM NaH2PO4 with 150 mM NaCl, 25% glycerol, 20 mM imidazole, 0.01% Triton X-100, at pH 8) using a Q-fill2 (Gentix, UK).
  9. Spin the filter plates
  10. Add 25 μL of elution buffer (50 mM NaH2PO4/150 mM NaCl/25% glycerol/250 mM imidazole/0.01% Triton X-100, pH 7.5)
  11. Incubate 15 min at 4 °C
  12. Elute proteins into 96-well receiver plates by centrifugation.
  13. Repeat 10-12
  14. Store the proteins in 96-well plates at -80 °C


10 hours


This work is supported in part by the National Institute of Health (GM071440 to C.H.; U54 RR020839 to H.Z.), W. M. Keck Foundation (to C.H.), the Arnold and Mabel Beckman Foundation (to C.H.), and the Research Corporation (to C.H.).

Associated Publications

A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli, Chien-Sheng Chen, Ekaterina Korobkova, Hao Chen, Jian Zhu, Xing Jian, Sheng-Ce Tao, Chuan He, and Heng Zhu, Nature Methods 5 (1) 69 - 74 16/12/2007 doi:10.1038/nmeth1148

Author information

Chien-Sheng Chen, National Taiwan Ocean University

Heng Zhu, Johns Hopkins University

Source: Protocol Exchange (2007) doi:10.1038/nprot.2007.544. Originally published online 19 December 2007.

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