Inclusion body prep

This generally takes me 7-9 hours, depending on how many tubes I'm processing at each sonication step.

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Buffers

RA: 100mM Tris pH 8.0, 5mM EDTA
R3: 100mM Tris pH 8.0, 0.5% Tergitol, 1% Triton X-100
R4: 100mM Tris pH 8.0, 1M NaCl
R5A: 50mM Tris pH 8.0, 6M GdnHCl

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Protocol

Day 1

Resuspend cell pellet in ~10ml aliquot of buffer RA. Optionally add 100ul PIC per 1g cells. I'm not sure if this is necessary, as proteins will be in inaccessible inclusion bodies, but haven't run controls.

Lyse by sonication: 80% with macrotip for 1min at 2s on / 1s off.
(Optional: degrade DNA with 40ul 10mg/ml DNase + 15ul 1M MgCl2 per 1g cells, leave at RT for 30mins)
Spin 35000g, 30 mins

Resusupend pellet in 6ml aliquot buffer R3 per 1g cells (Detergents)
Sonicate, 40%, 5x 10x 1s on/1s off pulses. Spin 35000g, 30min

Resuspend pellet in 6ml aliquot buffer R4 per 1g cells (High-salt)
Sonicate, 40%, 5x 10x 1s on/1s off pulses. Spin 35000g, 30min

Resuspend 1.5mL aliquot buffer R5A per 1g cells (Strong denaturant)
Sonicate 20%, 5x 10x 1s on/1s off pulses.
If necessary, add 7.5ul 1M DTT (mr = 154.25) per 1g original cells: i.e., 5mM DTT

Suggested modification: initially resuspend in a small amount of buffer, THEN add GdnHCl. This should greatly improve the resuspension and thus improve protein recovery. The IB pellet resuspends poorly (i.e., lumpily) into 6M GdnHCl immediately.

Incubate overnight in shaker at 4degC.

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Day 2

Sonicate again to remove all remaining solids.

Incubate another hour.

Centrifuge at 35000g for 30mins. Retain supernatant, throw out pellet.

Ultracentrifuge at 160000g for 30 mins. Retain supernatant, throw out pellet.
Filter as necessary and move on to the real science...



Old Day 2 method: Spin 35000g 30min to remove debris. After the centrifuge is at 35000g, flip the 'ARC' switch to enable a slow, controlled decceleration. Decant CAREFULLY! The pellet is liable to resuspend if you even think about shaking it. Experiment with pippeting instead of decanting, too.

Repeat 45000g spin multiple times, as necessary.

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morgan(at)mbi.ucla.edu