dspinella at chugaibio.com
Wed Sep 23 15:43:03 EST 1998
> As I am setting up a new project, I was wondering if the following method
> could be successful for isolating the cDNA of a particular plasma membrane
> 1. Transfect the complete cDNA bank in an appropriate acceptor cel
> 2. After a couple of days, coincubate the cell population with magnetic
> beads coated with an antibody against the protein and isolate adhering cells
> 3. Perform a Hirt-extraction on the isolated cells
> 4. Amplify the plasmid DNA in coli and transfect individual clones, using
> binding of the antibody as a screening method (maybe after a second or third
> "panning" round).
> Does anyone know if this has been applied with success in the past, and if
> there's any snag in this method?
> Many thanks,
As the previous respondent has pointed out, this is just a variant on
the widely used COS expression cloning technique pioneered by Brian Seed
(substituting magnetic beads for antibody-coated microtiter plates).
You can also use flow cytometry. Lots of genes encoding cell-surface
proteins have been cloned this way (including many CD molecules in
addition to CD2). Check the archives of this group for postings
pointing out the pitfalls -- its been covered here many times.
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