PURIFICATION OF HIS-TAGGED LUCIFERASE

Date: _____________

Cells collected on _______________

1. Resuspend pellet in 1 mL of EXTRACTION BUFFER (EB) for every 50 mL of culture harvested.
2. Add lysozyme to EB, prior to resuspending pellets, to a final concentration of 1 mg/mL
3. Incubate on ice for 30 min.
4. Vortex cells. Followed by 6 x 10 sec bursts of sonication with 10 sec cooling periods in between bursts.
5. Add RNAse A (10 µg/mL) and DNase I (5 µg/mL) and incubate on ice for 10-15 min.
6. Centrifuge lysate at 10,000 x g for 20-30 min at 4 C
7. Add ______ mL of 50% Ni-NTA slurry (equilibrated in EB). Mix gently at 4 C for 60 min.
8. Load column and wash with 10 bed volumes of WASH BUFFER #1
9. Wash with 5 bed volumes of WASH BUFFER #2
10. Elute with 5 x 1 bed volume of ELUTION BUFFER.
11. Remove 200 uL aliquot from each fraction for protein assays and store remaining fractions at -80 C.

 

BUFFERS

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EXTRACTION BUFFER:

50 mM NaH2PO4 pH 8.0

300 mM NaCl

20 mM Imidazole

20% (v/v) glycerol

Pefabloc

WASH BUFFER #1

50 mM NaH2PO4 pH 8.0

300 mM NaCl

40 mM Imidazole

20% glycerol

WASH BUFFER #2

50 mM NaH2PO4 pH 8.0

300 mM NaCl

60 mM Imidazole

20% glycerol

ELUTION BUFFER

50 mM NaH2PO4 pH 8.0

300 mM NaCl

250 mM Imidazole

20% glycerol