SURVEY response summary: GST-fusion elution
djt2 at po.cwru.edu
Fri Feb 10 12:45:46 EST 1995
In article <djt2-0902951216580001 at path22285.path.cwru.edu>,
djt2 at po.cwru.edu (Dennis Templeton) wrote:
> We and several of our neighbors have had problems with many GST-fusion
> proteins being very difficult to elute from the GST-sepharose. Several
> other of our friends say that it is no problem at all.
Here are the early responses we received (thanks everyone) and what I
think is a solution.
John (below) suggested keeping the pH high (pH 9.0) during the elution,
and this morning we tried that on a protein that had not eluted in 50 mM
GSH. Today, it eluted in 50 mM Tris pH 9 + 5 mM GSH. The pH answer is also
confirmed by a survey of my friends here locally; those of us who have
just tried elution in pH 7 PBS have had the failures. The source of the
beads seems irrelevant, and it was our 5 year old Sigma beads that worked
fine this morning.
My apologies to all if the pH issue is in the basic GST protocols, but it
seems that many of us have missed this point.
- - - - - - - -
> 1) Have you had problems with poor elution from GST-beads??
Yes. Most of our proteins are not eluted efficiently.
> 2) Did you come up with a means for good elution?
> 3) What Source were the beads that worked well?
> 4) What Source were the beads that did not elute well?
> 5) What do you think is going on? What is your evidence?
We have tried Phrmacia's and Molecular Probes' beads. Some proteins
were very efficiently recovered with those columns, but most were not.
It does not depend on the manufacturer or the lot of the beads. We
rather consider it depends on the nature of the protein. From our
experience, most of large fusions (>80kDa) do not bind well to the
beads and once bound, they cannot be eluted easily.
Yasushi Okada, MD.
($@2,ED9/;V(J <= Japanese Kanji)
Department of Anatomy & Cell Biology
University of Tokyo
($@El5~Bg0e!&Bh#12rK6(J <= Japanese Kanji)
Email:mm37301 at secc.ecc.u-tokyo.ac.jp
- - - - - - - - - -
In my hands GST not fused to anything elutes quite well with 5 mM GSH
(from Sigma). With anything more than a couple of kDa stuck on, 5 mM gave
very poor recovery -- when I boiled the beads in sample buffer and checked
on a gel, I found many times more protein than I had recovered with GSH.
Going up to 50 mM solved this problem for me. I'm sure it depends on the
particular protein you are studying. A more difficult problem (for me!)
which I have as yet been unable to solve is that several of my fusion
protein preps turn out to be largely truncated material, with only a small
fraction of the protein present as the full length fusion. I've tried
several protease deficient strains of E. coli but have seen no
improvement. Any ideas on how to deal with this?
LICHY at email.afip.osd.mil
- - - - - -
Anyway, I've generally had very good success eluting GST fusion proteins
from beads. These fusions are typically in the 85 -120 kDa range (getting
them soluble is another story). However, I had trouble eluting one of my
fusions until I realized that I had changed the elution conditions; on
this occasion, I was eluting in a modified PBS-type buffer at about pH
7.3. However, when I switched back to my normal elution buffer of 50 mM
Tris (pH 9.0!) + 20 mM glutathione that same fusion protein eluted
readily. So, I would suggest that another explanation might be that your
pH is marginally too low and is OK sometimes, but doesn't work other
times. Oh, and I've used both Pharmacia and Sigma beads with equal
rjl6n at uva.pcmail.virginia.edu
Institute of Pathology
CWRU School of Medicine
djt2 at po.cwru.edu
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