In article <8e1iro$5f5$1 at SonOfMaze.dpo.uab.edu>,
James F. George, Ph.D. <jgeorge at REMOVE_THISuab.edu> wrote:
>We have made extensive use of the Miltenyi system with good results for both
>depletion and enrichment experiments. The beads do not seem to interfere
>with functional assays or the ability of the cell types we are studying to
>mediate their function in vivo.
>>The largest drawback is, if you use their complete MACS system, it requires
>a fairly hefty up-front expenditure because the magnet is expensive.
Another alternative is the StemSep system by StemCell Technologies. The
up-front cost is less because the magnet is less expensive (although I
think the Miltenyi magnet is easier to use), and it is a little more
flexible because the Ab isn't directly linked to the beads. The beads are
coated with dextran, and the antibodies are biotinylated; a bifunctional
anti-biotin/anti-dextran antibody is then used to crosslink the two (this
might also avoid your steric hindrance problems). This allows the use of
a cocktail of antibodies for separation purposes, making negative
selection purification very efficient (I routinely do T cell preps of >95%
purity, and there are no beads bound to the T cells at the end). It also
allows you to use your favorite antibodies without having to covalently
link them to beads - all you need to do is biotinylate them.
Ken Frauwirth
--
Ken Frauwirth (MiSTie #33025) kfrauwir at midway.uchicago.edu
Abramson Cancer Research Institute
University of Pennsylvania
http://mail.med.upenn.edu/~kfrauwir
