Streptavidin DynaBeads for isolation of biotin tagged transmembrane
Austin P. So (Hae Jin)
who at what.where
Sun Sep 26 01:46:45 EST 2004
Arthur Beyder wrote:
> I have streptavidin DYnabeads that I can use to pull off the
> biotin-tagged protein. I've never done this before, does anyone have
> any suggestions or potential problems.
It would depend on how you biotin-tagged the ion-channel, and whether
you are concerned with pulling down any subunits or anchoring proteins.
But the dynabeads should work...the washing buffers are 1M NaCl (you
could add 0.1% SDS without losing anything), so that may or may not be a
problem. Also, getting it off the support will be a challenge, which is
why a cleavable biotin is worth considering.
Just in case you weren't aware, the Pierce and the Molecular Probes
catalogues are really good resources for everything biotinylated...
> Also, what would be the best way to find out whether I have enough
> protein to see it on a gel?
We used to use the BCA method (Pierce) to quantitate protein before
But let's say you can pick up femtomolar quantities (say via a
chemiluminescent method). If your ion channel (subunit?) is ~100 kDa,
then you need about 1e-15 x 1e5 ~ 100 pg.
Bottom line is that it really depends on what your expression levels are
(though I suppose that shouldn't be a problem since you are using HEK
cells), and how good your biotin-tagging method is.
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