In article <20030611235921.3792.qmail at ww02.hostica.com>, tkalog at lsuhsc.edu wrote:
>>I would like to get some ideas for exptl strategies that can be used to
> determine whether endocytosis of a given protein is required for its
> biological effect on cells. I already know this protein has cellular effects,
> I also know that it is endocytosed by adherent cells fairly quickly, in a
> saturable manner, and that about 30% of this uptake is specific. I need some
> approaches for testing a link between uptake and cellular effects. I've
> already got some ideas, and have some studies going using transfection of a
> dominant negative mutant for dynamin, but hopefully someone who has some
> experience with this can help with some other ideas--simple, complicated, old
> school or new technology, I don't care; at this point anything might help.
You can electroporate cells in the presense of [reasonably high]
concentrations of this protein - some of it will get into cells. (This was
use with neutralizing antibodies in the past). To eliminate endocytosis
at this point, you can do electroporation on ice and "shave" cells by
protease (and degrade your protein not entering the cells). Something
like pronase will do outstanding job although even trypsin should work
treat cells. Electropores seal to a very small diameter even on
ice within ~ 15 min for sure, so virtually no protein will be getting into
if added after electroporation. (Of course, inhibition by drugs is also an
option).
With proper controls, this will tell you if the cellular effects you
observe are dependent on edocytosis per se. If those effects require
that no dead cells were present (an unavoidable consequence of
electroporation), you can do cell sorting to separate alive from dead.
For this purpose, optimal electroporation conditions are going to be
different from DNA transfection.
If you, by any chance, need more info on this approach, email me
at k l e n c h i n <at> f a c s t a f f. w i s c . e d u.SMAMPROOF
(remove space and ..SPAMPROOF to reply)
DK
