I'm looking for a protocol of G protein activation in live HEK293 cells with AlF4-.
I'm trying to carry out irreversible activation of G proteins in HEK293 using AlF4-. I'm particularly interested in Gi/o family of G proteins. Unfortunately, my experiments were not successful up to now.
I normally move HEK293 cells to the room temperature, replace the medium with HEPES-buffered HBSS and apply freshly prepared AlCl3 (final concentration 30uM) and NaF (final concentration 10mM).
Is maintanance of +37C critical for this process? A single preliminary test with keeping cells at +37C didn't yeild positive result.
What is the timing of AlF4- activation (I found in the literature values between a few seconds to 20 minutes)?
If you happen to have a working protocol or any ideas what can be wrong with mine please tell me!