Purification

Purification:

1. Heat the suspended microsomes for 20 min at 65 °C.

2. Adjust the concentration of heated-microsomes to 2 mg ml^-1 with n-dodecyl-b-D-maltopyranoside (DDM) in a 1:3 ratio in solubilization buffer and heat for an additional 2 h at 65°C.

3. Immediately centrifuge at 4000 × g for 5 min at 4 °C. 

4. Collect the supernatants and cool them on ice for 10 min. 

5. Then centrifuge at 4000 × g for 20 min at 4 °C. 

6. Incubate the supernatants with Ni-NTA matrix for 1h at 37 °C after which the matrix is loaded into a column.

7. Wash the column with 10X column volume of washing buffer. 

8. Elute the protein with 3X column volume of elution buffer.

9. Dialyze the purified protein overnight against buffer (25 mM MES, pH 6.5, 3% glycerol, 120 mM NaCl, and 0.03% DDM). 

10. Subject it to size-exclusion chromatography using a Superdex^TM 200 Increase 10/300 GL column (GE Healthcare). 

Solutions:

1. Solubilization buffer: 

   50 mM MES, pH 6.5, 20% glycerol, 60 mM NaCl

   Washing buffer  
   25 mM MES, pH 6.5, 3% glycerol, 120 mM NaCl, 10 mM imidazole and 0.03% DDM

2. Elution buffer
   25 mM MES, pH 6.5, 3% glycerol, 120 mM NaCl, 500 mM imidazole and 0.03% DDM

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