Prbbbb:inclusion body solubilization v1
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.
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)
starting point: pellet of your insoluble fraction (after 40 min high-speed cold-room centrifugation of the lysate)
- add 500 µl buffer I to the pellet
- resuspend by pipetting or 2 times 5-10 s vortexing
- boil and load 5 µl + loading buffer on a SDS PAGE gel
- continue with purification using the same buffer without Triton and with varying amounts of Imidazole
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
raik:share your experience!
or instead, discuss this protocol.