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A story of Urea, dialysis and GST affinity purification... hopefully!

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#1 srr



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Posted 30 March 2009 - 08:45 PM

Hi everyone, Iím trying to isolate 3 different proteins that are expressed using the PGEX4t-1 vector within BL21 ecoli cells. My protein sizes range from 50 to 100kDa (with the gst tag). After expression all of my protein is found in inclusion bodies. I have tried a wide range of induction temperatures and times. I am hoping to attach the proteins to gst beads so that I can use them in pull down assays later on. I am now considering using urea to resolubilize the protein following which I will use sequential dialysis to help with refolding.

Does anyone out there have any success with inclusion bodies, urea, dialysis and GST affinity purification? If so what did you do and what buffers did you use?
I have a general protocol but any help or feedback would be greatly appreciated!

#2 Roo



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Posted 07 July 2009 - 12:12 PM

Many of the proteins I work on are insoluble (even after playing with induction and growth conditions). I used to go crazy trying to purify them, but years ago a neighboring lab that happened to be a protein lab gave me a copy of this article...An Improved Procedure for the Purification of Protein Fused with Glutathione S-Transferase
by Francesco Grieco, Joanne M. Hay, and Roger Hull
from BioTechniques Vol. 13, No. 6: pp 856-858 (Dec 1992).

I have used this method ever since with great success. It leaves out some of the early parameters on how to grow, induce, and start the purification, but we use commonly known techniques for that. We then resuspend the pellets in 5 ml/g pellet weight cold PBS + Proteasae Inhibitors and proceed with the conditions in the paper, but we recalculate the volumes by ratios of our pellet resuspension volume and their volumes. We then follow manufacturer's instructions for our GST bead binding, elutions, etc. Afterwards, we just dialyze into PBS or TBS.

I found this method to be much easier and less time consuming than the Urea methods, and the protein seems to work in most applications (binding/pull down assays, antibody production, etc.).

Good luck!

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