1. Resuspend pellet from lysozyme-treated & DNA-treated lysate in 50-ml buffer B (pH 7.6), 0.2 % octylglucoside.
2. Incubate 1 hour on ice with occasional mixing.
3. Pellet IBs at 10K fo 10 min in GSA rotor (4°C). Pellets from induced cells should pack nicely, & pellets from uninduced cells should be loose.
4. Resuspend in 50-ml buffer B. Store aliquots at -20°C. Determine concentration of tailspike chains by SDS-PAGE & densitometry.
1. Pellet inclusion body sample containing 50 µg tailspike chains (2 min at 14K & 4°C in microfuge). Aspirate supernatant.
2. Dissolve pellet in 6 M urea, 10 mM DTT, 50 mM Tris-HCl (7.6), 25 mM NaCl, 2 mM EDTA. Pellet dissolution may require pumping several times with P200.
3. Incubate for 1 hour at RT. Vortex occasionally.
4. Remove insoluble material (5 min at 14K & 4°C).
5. Chill supernatant on ice & dilute 10-fold with ice-cold buffer B.
6. Shift to 20°C after 1 hour on ice.