Prbbbb:inclusion body solubilization v1: Difference between revisions
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(New page: 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 c...) |
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Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! | Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! | ||
<font face="courier"><nowiki>raik:</nowiki></font> | <font face="courier"><nowiki>raik:</nowiki></font>share your experience! | ||
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Revision as of 02:10, 26 August 2009
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:
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)
- 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
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
raik:share your experience!
References
Contact
or instead, discuss this protocol.
