Helen,
I heartily agree with everything Tom says below. Serum Free Media is the
way to go if you can. You are likely to still have albumin issues, but they
are probably an order of magnitude less than with serum based media. Dye
matrixes do remove albumin, but not absolutely. Cibicron blue, as has been
mentioned before, works OK and probably as good as anything. I have used
some matrixes a long time ago that were reportedly designed for "albumin
removal" and they did not work any better than did cibicron blue.
Albumin, as you are learning, is really hard to get rid of. A while back, I
did some mass spec identification of a soluble protein isolated from human
fibroblasts grown in serum free media. I cut bands out of a 1-D SDS-PAGE
gel. In the four bands submitted for MS, each band contained a fair bit of
albumin, in spite of the fact that each band was at a molecular weight of
much greater than 66 kDa. Basically, the stuff hangs on to everything in
even the harshest conditions.
Jim Lawrence
Pioneer Hi-Bred, Intl.
Johnston, IA 50131
515-270-5909
-----Original Message-----
From: Thomas Andersen [mailto:AndersT@mail.amc.edu]
Sent: Monday, February 19, 2001 8:23 AM
To: Recipients of ABRF List
Subject: Re: Blue-Sepharose
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.
This archive was generated by hypermail 2b29 : Fri Feb 23 2001 - 13:03:34 EST