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Solubilization and renaturation of proteins from inclusion bodies Solubilization and renaturation of proteins from inclusion bodies


Materials

Wash buffer

50 mM Tris-HCl pH 7.5

50-200 mM NaCl


Extraction buffer

50 mM Tris-HCl pH 7.5

8 M urea

1 mM DTT

1 mM PMSF*

*
 PMSF is instable in aqeous solutions and added to the buffer atthe point described in the protocol.


Stock solutions

100 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol.



Procedure


1. Centrifuge the cell lysate at 4°C for 20 min at 30 000 g [16 000 rpm using a SS-34 rotor (Sorvall)].
2. Resuspend the pellet in 10 ml wash buffer containing 1% Triton X-10 and 1 M urea per gram cell wet weight. Incubate at room temperature for 5 min.
3. Centrifuge the cell lysate at 4°C for 20 min at 30 000 g [16 000 rpm using an SS-34 rotor (Sorvall)].
4. Repeat step 3 twice.
5. Resuspend the pellet in 10 ml wash buffer and centrifuge the suspension at 4°C for 30 min at 45 000 rpm using a 45Ti rotor (Beckman).
6. Resuspend the inclusion bodies in extraction buffer. Use so much buffer that the final protein concentration in the solution is 1 mg/ml.
  • Add 10 µl PMSF (100 mM) per ml of solution at this point.

Incubate the solution at room temperature for 60 min.
7. Dilute the solution tenfold with extraction buffer (final protein concentration is 0.1 mg/ml) and dialyze overnight against a 100 fold volume of wash buffer.
8. Centrifuge the dialysate at 4°C for 30 min at 45 000 rpm using a 45Ti rotor (Beckman).