Streptavidin DynaBeads for isolation of biotin tagged transmembrane protein

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 
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



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