Hi Juliana,
Since you can purify some protein with IMAC, I suggest that you do a
buffer screening with Thermofluor before running the desalting column
(or gel filtration column) and use the buffer condition(s) that
stabilise your protein for the next purification step and possibly
freezin
An added benefit of EDTA is that it inhibits some proteases - for one
of our wimpier proteins, spiking each fraction collector tube with a
little EDTA before running the Ni column really helped reduce keep
the sample in one piece.
Phoebe
At 01:18 PM 2/19/2008, Sophia Tsai wrote:
Hi,
Hi,
Agreed on this. I used to have issues with aggregation due to the Nickel
being stripped off the column (happens with elution in imidazole). Adding
10mM EDTA to the elution immediately AFTER it has come out of the column
will chelate the Ni++ and prevents aggregation (at least in my case).
Afte
Hi,
One has to bear in mind that adsorption at high pH (above 6.5 is already
high for Imac; pKa of exposed His is 6.2) leads to stronger interactions
which, in turn, require stronger eluting conditions, e.g. > 200 mM
imidazole (pKa ~ 7.0).
I suggest lowering the pH of the adsortion buffer, e.g
Hi,
I used another way to deal with this problem. You can try to elute your
protein with the buffer containing 50mM EDTA (You need at least 10CV to
elute completely). Then use gel filtration to remove the Ni.
I applied this method with two proteins and it showed good results.
Good luck!
TriNgo
--
Hi,
In addition to the tips already suggested, you could also try to elute
your protein with histidine.. I've got a protein that crashes out with
imidazole as well, and I have succesfully used 200mM histidine for
elution (using either KPi or MES buffer in my case).
Cheers,
Ronnie
On Feb
Firstly - please forgive my last 'blank' message - comes of trying to
e-mail whilst travelling on a train!
I would suggest that you change the affinity tag that you're purifying
with. Go with either a GST or Strep-tag. That way you are avoiding the
imidazole that is obviously causing problems wi
--
Dr Antony W Oliver
Cancer Research UK: DNA Repair Enzymes Group
Section of Structural Biology
The Institute of Cancer Research
237 Fulham Road
LONDON SW3 6JB
[EMAIL PROTECTED]
020 7153 5488
--
>>> Juergen Bosch <[EMAIL PROTECTED]> 02/15/08 5:23 PM >>>
Not sure if somebody mentioned it already,
Not sure if somebody mentioned it already, but have you played with
other buffers than the recommended sodium phosphate from the manual ?
Most lazy people use Tris because there's a 1 M stock solution - but you
can try also other buffers as long as you stay in a pH range >pH6 Of course you sti
Dear all,
I am with the same problem of Jacob.
My protein is
precipitating, especially when I 'freeze it'. I am using a His Trap HP column
coupled with a desalting column. Already tried to elute with gradient of
imidazole (that allowed an elution with less amount of imidazole), but
even then, t
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