Prbbbb:inclusion body solubilization v1

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Current revision (04:13, 26 August 2009) (view source)
(Procedure)
 
Line 27: Line 27:
# 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==

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Contents

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!

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References

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