Purification of recombinant bacterial proteins

Ni-NTA agarose bead (Qiagen, 30210)

IPTG

Triton X-100

LB/Kan media

Lysis Buffer (see Recipes)

Wash Buffer (see Recipes)

Elution Buffer (see Recipes)

1. Grow culture in 50 mL LB/Kan media until it reaches OD= 0.4 to 0.6. Add IPTG to its final concentration of 0.4 mM and induce 6x His-tagged protein production for 2 h at 37°C.
2. Cultures were centrifuged at 5,000 rpm for 15 min at 4°C and lysed in 1×10⁷ cell/25 μL lysis buffer for 30 min at 4°C.
3. Sonicate cells on ice twice for 1 min with 10 second break. After sonication, add 1% Triton-X into lysate.
   Setting: Amplitude 50%, 4 pulses. (The sonicator probe should go under the lysate surface to avoid foaming). 
4. Centrifuge lysate 20 min at 12,000 rpm at 4°C to pellet cellular debris. 
5. Transfer the supernatant in to 15 mL falcon tube. 
6. Add 500 μL of Ni²⁺-NTA-agarose beads pre-equilibrate with lysis buffer (take 1 mL of Ni²⁺-NTA-agarose bead and centrifuge 1 min at 3,000 rpm, discard the supernatant and wash with 1 mL of lysis buffer three times) and mix gently by rotating for overnight at 4°C.
7. Beads were washed with 1 mL washing buffer at RT incubating 3 min.
8. Elute the recombinant protein by incubating beads in 500 μL of elution buffer for 20 min at RT with gentle agitation.
9. Centrifuge the beads 5 min at 3,000 rpm at and transfer supernatant in to 1.5 mL Eppendorf tube.

Recipe 

1. Lysis buffer
   50 mM NaH₂PO₄
   300 mM NaCl
   10 mM imidazole
   1 mM PMSF protease inhibitor 
2. Wash Buffer
   50 mM NaH₂PO₄
   300 mM NaCl
   20 mM imidazole
   1 mM PMSF protease inhibitor 
3. Elution buffer
   50 mM NaH₂PO₄
   300 mM NaCl
   250 mM imidazole
   1 mM PMSF protease inhibitor 

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