Isolating Proteins from Inclusion Bodies

Adapted from Antibodies – A Laboratory Manual ch.5, pg. 90

Dana L. Miller

8 Nov 1999

1. Resuspend cell pellet in 100mM NaCl, 1mM EDTA, 50mM Tris-HCl pH 8.0 to a final concentration of 10% (v/v).
2. Add both DTT and PMSF to a final concentration of 1mM.
3. Sonicate cells until they are well broken up (OD₆₀₀ is 1/10 of its original density) without allowing the cell suspension to foam.
4. Remove cell debris by centrifugation at 10,000 x g for 10 min at 4˚C.
5. Wash each pellet with ice-cold 100mM NaCl, 1mM EDTA, 50mM Tris-HCl pH 8.0, 0.1% sodium desoxycholate.
6. Incubate on ice for 10min with occasional mixing.
7. Add MgCl₂ to a final concentration of 8mM (200μL 1M MgCl₂/25mL cells) and DNase to a final concentration of 10μg/mL.
8. Incubate on ice with occasional mixing for 30-60mins.
9. Remove inclusion bodies by centrifuging for 10 min at 10,000 x g at 4˚C. Slower speeds may be suitable and could reduce the background.
10. Wash pellet in same volume of 1% Igepal CAG30, 100mM NaCl, 1mM EDTA, 50mM Tris-HCl pH 8.0.
11. Centrifuge for 10min at 10,000 x g.
12. Wash pellet in 1M urea, 50mM Tris-HCl pH 8.0 and centrifuge 10 min at 10,000 x g.
13. Resuspend final pellet in desired amount of 8M urea, 100mM NaCl, 1mM EDTA 50mM Tris-HCl pH 8.0 and 1mM DTT.