Prbbbb:inclusion body solubilization v1

Back to all protocols

Overview
This protocol describes how to re-dissolve the insoluble fraction of an E. coli protein expression for purification on His-Trap columns. It picks up where the expression protocol ends.

See also:
 * Drummond lab yeast protocol
 * Knight lab bacterial protocol

Buffers
buffer I (for purification on His-Trap columns)


 * 50 mM HEPES (pH 7.4)
 * 0.5 M NaCl (high salt)
 * 5 mM DTT (reducing conditions)
 * 8 M Urea (chaotropic, unfold proteins)
 * 1% v/v Triton X-100 (detergent, help solubilization of hydrophobic peptides)
 * 20 mM Imidazole (for reducing unspecific binding on the column)

Procedure
starting point: pellet of your insoluble fraction (after 40 min high-speed cold-room centrifugation of the lysate)


 * 1) add 500 µl buffer I to the pellet
 * 2) resuspend by pipetting or 2 times 5-10 s vortexing
 * 3) boil and load 5 µl + loading buffer on a SDS PAGE gel
 * 4) continue with purification using the same buffer without Triton and with varying amounts of Imidazole

Contact

 * Raik

or instead, discuss this protocol.