Dear Fred --
just to check... are you sure you have the His-tag? Might have been
cleaved somehow? You might want to increase the number of His in the
tag as well.
HTH.
-- Leo --
On 27 Jan 2009, at 21:00, Fred wrote:
Hi ccp4 list,
I am trying to purify a his-tag protein by metal
am *Di, 27.1.2009:
*
*Von: Fred ccp4bb.l...@gmail.com
Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
An: CCP4BB@JISCMAIL.AC.UK
Datum: Dienstag, 27. Januar 2009, 22:00
*
*Hi ccp4 list,
I am trying to purify a his-tag protein by metal affinity chromatography
ccp4bb.l...@gmail.com
Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
An: CCP4BB@JISCMAIL.AC.UK
Datum: Dienstag, 27. Januar 2009, 22:00
*
*Hi ccp4 list,
I am trying to purify a his-tag protein by metal affinity
chromatography. The
protein was expressed in inclusion
Talon and
let you know the results.
All the Best,
Fred
--- Fred /ccp4bb.l...@gmail.com/ schrieb am *Di, 27.1.2009:
*
*Von: Fred ccp4bb.l...@gmail.com
Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
An: CCP4BB@JISCMAIL.AC.UK
Datum: Dienstag, 27. Januar 2009, 22:00
28, 2009 9:08 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
Just to let you know. No way, Talon also don't work. I am gonna try the
GE His-trap column.
Fred wrote:
Hi everyone,
Thanks for answer my question. Just to add some more notes regarding
to my
-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Fred
Sent: Wednesday, January 28, 2009 12:08 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
Just to let you know. No way, Talon also don't work. I am gonna try the
GE His-trap column
Hi ccp4 list,
I am trying to purify a his-tag protein by metal affinity
chromatography. The protein was expressed in inclusion bodies and its
his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea
8M or GndHCl 6M). Playing with NaCl and detergents didn't help much.
Any help is
Hi Fred,
I used to worked with a protein which would not bind to the Qiagen
NiNTA (really *NOT*) but beautifully to IDA resin (e.g. Amersham
Pharmacia).
This was under non-denaturing conditions, though, and I don't know whether
this applies to the denatured state. It may be worth a try.
You can also try TALON cobalt affinity resin from Clontech (
http://www.clontech.com/products/detail.asp?product_id=10588tabno=2) or the
high yield PrepEase resin from USB (
http://www.usbweb.com/category.asp?special=cat=234id=78806).
On Tue, Jan 27, 2009 at 4:34 PM, Tim Gruene
You don't have EDTA left over from your protease inhibitor, do you?
Some commercial cocktails include it. You may have to read the fine
print.
James
On Jan 27, 2009, at 1:00 PM, Fred wrote:
Hi ccp4 list,
I am trying to purify a his-tag protein by metal affinity
chromatography. The
To go along with the EDTA comment, did you check the pH of your binding
solution? Sometimes lysing E. coli causes the pH to drop below the pKa of
the histidines.
On Tue, Jan 27, 2009 at 8:10 PM, James Stroud xtald...@gmail.com wrote:
You don't have EDTA left over from your protease inhibitor,
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