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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

Native Purification of His-Tagged Proteins From Sf9 Cells Using Ni-NTA

Native Purification of His-Tagged Proteins From Sf9 Cells Using Ni-NTA
 
Procedure
1. Centrifuge cells at 700 X g in a table top centrifuge or equivalent and remove the supernatant. Wash the cell pellet once by resuspending it in 10 ml of ice-cold PBS and repellet the cells.

2. Resuspend the pellet in 20 ml of Lysis Buffer.

3. Transfer the solution to an oakridge tube and lyse the cells by sonication using a Polytron sonicator at a setting of 4 for 20 sec.

4. Centrifuge the lysate in an SS34 rotor for 20 min at 10,000 rpm (12,000 X g) at 4°C.

5. Pre-equilibrate 0.5 ml of Ni-NTA beads by suspending it in Lysis Buffer.

6. Add the lysate supernatant to the pre-equilibrated Ni-NTA beads. Rock at 4°C for 1 hr to overnight.

7. Transfer the lysate/Ni-NTA slurry to a Biorad econocolumn and collect the flow-through.

8. Run 20 ml of Wash Buffer 1 through the Ni-NTA column.

9. Run 20 ml of Wash Buffer 2 through the Ni-NTA column.

10. Elute the His-tagged proteins from the Ni-NTA by running 5 ml of Elution Buffer through the column, collecting 1 ml fractions.

11. Pool the first two fractions and dialyze overnight against 2 liters of the desired buffer.

Solutions
PBS   pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
Lysis Buffer   1 ug/ml Leupeptin
CAUTION! see Hint #2.
50 mM Tris, pH 8.0
1 mM PMSF
1 % (v/v) NP-40
Elution buffer   1 mM PMSF (CAUTION! see Hint #2)
500 mM NaCl
50 mM Tris, pH 8.0
1 M Imidazole
Wash buffer 2   1 mM PMSF (CAUTION! see Hint #2)
500 mM NaCl
5 mM Imidazole
50 mM Tris, pH 8.0
Wash Buffer 1   1 mM PMSF (CAUTION! see Hint #2)
500 mM NaCl
50 mM Tris, pH 8.0
 
BioReagents and Chemicals
Imidazole
Leupeptin
PMSF
Tris
Potassium Chloride
NP-40
Sodium Chloride
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
 
Protocol Hints
1. This protocol is designed for processing 100 million cells.

2. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.