Difference between revisions of "Prbbbb:inclusion body solubilization v1"

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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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# add 500 µl buffer I to the pellet
 
# add 500 µl buffer I to the pellet
 
# resuspend by pipetting or 2 times 5-10 s vortexing  
 
# resuspend by pipetting or 2 times 5-10 s vortexing  
 
 
# boil and load 5 µl + loading buffer on a SDS PAGE gel
 
# 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
  
 
==Notes==
 
==Notes==
 
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!
We are just starting to use this protocol -- share your experience!
 
  
 
<!--Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.-->
 
<!--Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.-->

Latest revision as of 01:13, 26 August 2009

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:

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

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.