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
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
raik: We are just starting to use this protocol -- share your experience!
or instead, discuss this protocol.