Re: [ccp4bb] Glutathione rsin

[Barbara Giabbai]

Hi all.
 I don't have any experience with Clontech and Fisher 
resins, but about the GE one I faced the same problem as 
Sebastiano indicated.
But I have to say that the problem was not general, but 
protein (or family of protein) related: the low binding 
depends on the oligomerization state of the chimeric 
protein. When it behaves as dimer (GST dimerizes) it's 
fine, when the oligomeric state increases (in my case 
hexamer) I really can't manage to purify the protein on 
GST sepharose. GST not exposed...I guess...


I'm waiting for the replies Mirek's question...I'm 
interested too.


Ciao,
 Barbara




On Wed, 5 Jun 2013 09:32:28 +0200
 Sebastiano Pasqualato sebastiano.pasqual...@gmail.com 
wrote:


Hi Mirek, hi all,
I'm also very interested in the topic, so please keep me 
up with the replies, or make sure to post a summary, 
please.


In addition to the price, the problem we're facing with 
GSH-beads from GE (although we haven't tried others yet) 
is that we can't manage to deplete our lysates.
We are always left with a large amount of unbound 
GST-tagged protein in the flow through, that is 
eventually captured by a second, third and sometime 
fourth  incubation with fresh beads.

Using larger beads volume won't help.
Has anybody faced and/or overcame this problem?

Thanks a lot in advance,
ciao,

Sebastiano


On Jun 4, 2013, at 8:54 PM, Cygler, Miroslaw 
miroslaw.cyg...@usask.ca wrote:



Hi,
I would like to ask the bb faithful for their experience 
with the glutathione affinity resins. We have been using 
so far the Glutathione Sepharose fast flow from GE but 
the price is getting steeper. We found Glutathione 
Superflow resin from Clontech to be significantly less 
expensive and Glutathione agarose from Fisher somewhere 
in-between. We have no experience with the latter two 
resins and I wonder what is the experience of other 
people with these resins? Do they have decent binding 
capacity? Can they be efficiently regenerated or are they 
a single use only?

Thanks for your help,

Mirek





--
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990








Barbara Giabbai

Elettra-Sincrotrone Trieste S.C.p.A.
SS 14 - km 163,5 AREA Science Park
34149 Basovizza, Trieste ITALY
Office:   +39 040 375 8840
Lab:  +39 040 375 8537

Re: [ccp4bb] cell disruptor / homogenizer

[Barbara Giabbai]

Dear Clemens,
 I used the Panda homegenizer (GEA Niro Soavi) to 
disrupt E.coli cells: is really good, I always did the 
maintenance on my own, is not very expensive and easy to 
use. I found bigger models also in the big pharmas...so is 
really reliable.


http://www.niro-soavi.com/home.html

Hope to be helpful.

Cheers,
 Barbara


On Mon, 25 Mar 2013 10:16:55 +0100
 Clemens Steegborn clemens.steegb...@ruhr-uni-bochum.de 
wrote:



Dear colleagues,



We are currently looking for new cell 
disruptor/homogenizer equipment,
mainly for E.coli work. 




We currently have a Branson sonifier and a Microfluidics 
Fluidizer - the
latter one keeps causing trouble, and we think about a 
replacement.


We previously also used an Avestin C-5 Emulsiflex, 
required quite some
maintenance, and I inquired about a Constant Systems TS 
0.75 and got mixed
responses from users (not to mention their outrageous 
pricing).  




My question: 

What is your experience with Fluidizer, Emulsiflex, TS 
0.75?


Is there any other great (low maintenance, affordable - 
especially
concerning follow-up costs: repairs, replacement parts) 
equipment I missed? 




Thanks in advance for your comments



Best regards

Clemens





Barbara Giabbai

Elettra-Sincrotrone Trieste S.C.p.A.
SS 14 - km 163,5 AREA Science Park
34149 Basovizza, Trieste ITALY
Office:   +39 040 375 8840
Lab:  +39 040 375 8537