Hi Fellows,
my lab mates successfully expressed a glycoprotein in CHO cells in serum
free medium, and
the protein captures nicely on HisTrap Excel 1ml columns (obviously, high
yield is not my problem.).
We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole
gradient. 20mM
: [ccp4bb] HisTrap Trap
Hi Fellows,
my lab mates successfully expressed a glycoprotein in CHO cells in serum free
medium, and
the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield
is not my problem...).
We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep
Hi Bernhard,
I just stumbled over this patent, where they add cobalt or nickel ions:
http://www.google.com/patents/WO2013082518A1?cl=en
[0086] Supplementing cell culture media, such as CD FortiCHO and
Freestyle 293, with metal ions does prevent column stripping and improve
histidine tagged
2014 14:38:12 +
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf
of Keller, Jacob kell...@janelia.hhmi.org)
Subject: Re: [ccp4bb] HisTrap Trap
To: CCP4BB@JISCMAIL.AC.UK
Well, this is of only possible relevance, but in a
previous lab, we used sf9 cells/media quite a bit
Bernhard,
We have had similar, but not identical issues with some insect cell media, as
well as column interference by lipid. If we see this, we tend to run all the
material with the protein (cell lysate or media) through step-elution ion
exchange (quick on, quick off). While the
...@xs4all.nl]
Sent: Montag, 19. Mai 2014 16:55
To: b...@hofkristallamt.org
Cc: ccp4bb@jiscmail.ac.uk
Subject: Re: [ccp4bb] HisTrap Trap
Hi Bernhard,
I just stumbled over this patent, where they add cobalt or nickel ions:
http://www.google.com/patents/WO2013082518A1?cl=en
[0086] Supplementing
Bernhard,
One other point, after rereading your email. For step-elution ion exchange
(quick on, quick off), you can use some very cheap and quite robust media the
will resist a lot of stuff. Have them pour their own columns and toss it away
when it gets too dirty. You don't have to use the
with metals if you have a
machine handy---it's just an hour or so.
JPK
*From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf
Of *Bernhard Rupp
*Sent:* Monday, May 19, 2014 10:14 AM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] HisTrap Trap
Hi Fellows,
my lab mates successfully
: Re: [ccp4bb] HisTrap Trap
I think the new versions of GE's HisTrap columns can address these problems.
Try contacting someone at GE.
I think a number of labs have had such problems in the past and the culprit
has been believed to be a chelator in the media but never confirmed because
...@medimmune.com]
Sent: Montag, 19. Mai 2014 17:38
To: b...@hofkristallamt.org
Subject: RE: [ccp4bb] HisTrap Trap
Orange to pink colored media is a sign of phenol presence which is added to
buffer. That phenol is the problem. I was having similar problem and asked
not to add it. Everything worked well
I worked for several years on mammalian cell expression of extra-cellular
domains of single spanning membrane proteins
and other membrane proteins.
Just like you did when we switched stable lines from serum containing to
serum free medium the expression drastically
reduced.
So after switching to
by some metal ions from the medium.
(Loading 1L medium to a 1mL column does not sound like a great idea to me
anyways...)
Zhijie
From: Bernhard Rupp
Sent: Monday, May 19, 2014 10:13 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] HisTrap Trap
Hi Fellows,
my lab mates successfully
.
(Loading 1L medium to a 1mL column does not sound like a great idea to me
anyways...)
Zhijie
From: Bernhard Ruppmailto:hofkristall...@gmail.com
Sent: Monday, May 19, 2014 10:13 AM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] HisTrap Trap
Hi Fellows,
my lab mates
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