Hi Jacob,
Two points which I don't think others have mentioned:
Have you checked the pH of your imidazole (or pHed the solution before
elution)? It may be that the imidazole has driven up your pH, causing
protein to crash out.
You can add various things to fraction tubes prior to elution so t
Dear all, thank you so much for all the invaluable tips that were delivered
faster than FedEx (and sorry for not being able to reply to individual
emails). My problem was resolved by adding EDTA (to about 5 mM), spinning
down at 15K for 10min to get rid of precipitation (I did have some even with
t
riday, February 15, 2008 9:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] rescuing crashing-out protein eluted from Nickel
column
Hi,
I also encounterd the same kind of problem with my protein. I noticed
that the protein eluted from Ni-NTA with 150mM imidazole or 300mM didn't
behave i
Jacob,
This is not a uncommon problem with Ni-chelation chromatography (on
Ni-NTA or Talon) with soluble and membrane proteins. In most of my
experiences, it is a salt problem (i.e., too little), but
hydrophobicity issues abound, as well.
On a pilot scale, just add any of the suggested a
this compound was very cheap).
best wishes,
Mathews
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Wong
Sent: Thursday, February 14, 2008 4:26 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] rescuing crashing-out protein eluted from Nickel
cuing crashing-out protein eluted from Nickel
column
Dear all,
I just ran into this problem and would like to see if I could get some
helpful tips before my protein completely crashes out.
I have a protein as 6His fusion and it remained bound to the Ni resin
with 40 mM Imidazole wash (added to
Artem
_
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob
Wong
Sent: Thursday, February 14, 2008 7:26 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] rescuing crashing-out protein eluted from Nickel column
Dear all,
I just ran into this problem and would like to see if I
first aid panic rescue might be to quickly add more salt.
(e.g. in couple of steps to 0.5 M-1 M NaCl and see if it clears up).
then add EDTA/or/and dialyse. (have to get teh salt out probably...)
the Ni-ions leaking out are a likely cause. among others.
tommi
Quoting José Trincão <[EMAIL P
Hi Jacob,
try adding a bit of edta (1mM) to remove any Ni that might come off the
column. You could also add some DTT or bME to keep cysteines reduced
(careful, add only after the edta!). In my experience the gradient did
not work very weel because the protein with have a lot of impurities.
Yo
Thank you all for the quick replies - I'm trying many of the tips in small
batches and would send out a feedback as soon as appropriate, gratefully, -
J.J.
On 2/14/08, Jacob Wong <[EMAIL PROTECTED]> wrote:
>
> Dear all,
>
> I just ran into this problem and would like to see if I could get some
> h
M
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] rescuing crashing-out protein eluted from Nickel column
Dear all,
I just ran into this problem and would like to see if I could get some
helpful tips before my protein completely crashes out.
I have a protein as 6His fusion and it remained bound to
Dear all,
I just ran into this problem and would like to see if I could get some
helpful tips before my protein completely crashes out.
I have a protein as 6His fusion and it remained bound to the Ni resin with
40 mM Imidazole wash (added to 1XPBS) but then was eluted off with 200 mM
(added to 1XP
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