Streptavidin DynaBeads for isolation of biotin tagged transmembraneprotein

Hello,

I would like to try to extract a biotin-tagged voltage-gated ion
channels from transfected HEK cells (alternatively I could use C6 or
astrocytes, but I prefer HEKs). I have no experience in biochemistry, I
apologize for oversimplifying the problem.

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.

Also, what would be the best way to find out whether I have enough
protein to see it on a gel? This is perhaps my biggest concern, I'd
like to be sure that there is enough sample.

Thank you,
Arthur

Arthur Beyder wrote:


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...


We used to use the BCA method (Pierce) to quantitate protein before
loading.

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.

Austin