Prbbbb:inclusion body solubilization v1

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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

1. boil and load 5 µl + loading buffer on a SDS PAGE gel

Notes

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