Helen
we have used Cibacron Blue ( Sigma) to deplete albumin prior to 2 DE with a
certain amount of success: the process certainly does not remove all
albumin but it reduces it to the extent that there is an increase in the
resolution of other less abundant proteins.
Suggested buffers are 50mM Phosphate, neutral pH or We use 50mM Tris, 50mM
NaCl, pH 7.2. There are some reports that suggest that tris shows decreased
albumin binding relative to phosphate, so you might want to try 50mM phosphate.
Depletion can be done either by batch binding in a microfuge tube or by
packing a small column in a pipette tip. Bed:sample ratio needs to be as
large as possible: suggest 3:1 v:v
Something to be aware of: there is a certain amount of non specific binding
to other plasma proteins.
Good luck
Derek
At 16:42 16/02/01 -0600, Helen Kim, Ph.D. wrote:
>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.
Derek Van Dyk
Australian Proteome Analysis Facility
Building F7B
Macquarie University
NSW 2109
Australia
http://www.proteome.org.au/
Ph: 61-2-9850 6205
Fax 61-2-9850 6200
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