Mini centrifuge filters for protein binding studies
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Mon Nov 15 16:06:07 EST 1999
:I would like to use mini centrifuge filters for studies of small radioactive
:ligands binding to a purified protein. At present I have a pressure cell,
:with eight chambers, which uses nitrogen gas pressure to force samples
:through 10,000 MW (Amicon TM10) filters; however, this is rather cumbersome
:Commercial filter units are available with 10,000 MW cut-offs; however, these
:are intended for _concentrating_ small protein samples. They are constructed
:so that the solution being filtered will not all pass through the filter.
:(They have a "retentate volume" of 10 microlitres.)
:What I need are minicentrifuge tubes with zero retentate volume (i.e. all
:the solution passes through the filter and the protein is left behind with
:bound radioactive ligand.) The total volume of my binding reactions is 100
:microlitres, so a 1.5 mL centrifuge tube would be a good size. Does anyone
:know where to obtain these? I am trying to make some from various 1.5 mL
:and 0.5 mL tubes, but it is tricky.
The standard technique used for this purpose for decades
involves protein/ligand filtration through regular 0.45 micron
pure nitrocellulose filters. Practically all proteins are retained
quantitatively on them, and only few LMW ligands bind.
I've done tons of these assays measuring GTPgS binding to
G-proteins and during rhodopsin phosphorylation assays.
Best filters are from Whatman and the most convenient
filtration manifold is from Hoefer/Pharmacia. Also allows
to collect ligand solution that goes through. In case you
have high backgrounds, you'd need to wash the filters
by filtration and the technique becomes non-equilibrium
(unless binding is _very_ strong). But so is filtration
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