I've only just tagged on to this thread, so I'm using Thomas's reply as a
foot hold - sorry Thomas!
We've routinely used strong anion exchanger resin to remove albumin from
tissue culture supernatant prior to purification, so maybe this will help
with your problem. We adjust the super. to pH 8 with Tris base and run
over Q-Sepharose equilibrated at pH 8 with 50mM Tris buffer. Admittedly
our proteins generally have pI ~9, so they flow right through, leaving the
albumin bound to the column, but maybe it will help....
Cheers,
Roger
At 01:22 AM 2/20/01 +1100, Thomas Andersen wrote:
>Helen-
>
>I agree with Derek's assessment that blue sepharose is good to 'deplete'
>albumin, but not eliminate it. It often takes more than one pass over a
>column. Especially since you refer to conditioned media as the starting
>material. Media is often supplemented with 5 or even 10% fetal calf
>serum, which contains a "ton" of albumin. To deal with this, we
>actually switched the cells to serum-free media for their last several
>hours before examining media for our protein of interest, and still
>found it necessary to use more than one pass over blue sepharose. Blue
>sepharose does work, but it can't work miracles in the face of
>mega-quantities of albumin.
>
>Tom
>
>
>
>____________________________
>Thomas T. Andersen, Ph.D.
>Assistant Dean
>Albany Medical College
>Albany, NY 12208
>
>518 262-5253
>518 262-5183 fax
>anderst@mail.amc.edu
>
>
> >>> "Helen Kim, Ph.D." <Helen.Kim@ccc.uab.edu> 02/16/01 05:42PM >>>
>Hi All,
> Has anyone had experience removing albumin with Blue-sepharose?
>I
>know in principle it can be done, and I've read lots of papers where
>people
>blithely say they did with the blue sepharose. I recommended the
>procedure to a grad student dealing with blob of albumin in his 2-D
>gels
>of conditioned media. He bought some, tried it (I don't know exactly
>what
>he did), and he says "it didn't work." He's very bright, though, so I
>trust
>he went through reasonable efforts, but I thought I would check with
>you
>all, since there's no point re-inventing the wheel.
>
>
>
------------------------------------------------------------
Roger Murphy, Ph.D.
Biological Production Facility
Ludwig Institute for Cancer Research
Austin & Repatriation Medical Centre
Studley Road,
Heidelberg, Vic. 3084
Australia.
Tel 61-3-94965463
Fax 61-3-94965436
Email Roger.Murphy@Ludwig.edu.au
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