Ric3 NAChR 7 expression in HEK cells - (Mar/07/2013 )

Hi all,

I have a big problem expressing Ric3 NAChR 7 in HEK cells. It should be easy but I can't get it working at all.
I transfect in 12 well plates with 2.5 e5 cells per well with 2 to 2.5 microliter of lipofectamine 2000. Cells are plated with 10% FBS. The lipofectamine and vectors are mixed with medium without FBS: both 5 min in medium and then I mix the DNA and lipo and leave for 20 min and carefully put on the cells.
I use 3 vector:
-EGFP , 1/10 to 1/100 --> I always have some nice green cells , so tranfection is working
- Commercial (origene) RIC3 and alfa 7 vectors, diluted in water ....tried even without maxiprep, so not contamination is expected.
total amount of DNA per well: from 1 to 2 microgram

In the literature you find almost all ratios between ric 3 and alfa 7 :
- 12:1
-1:1
-1: 500 !

I tried almost everything : no current measured with patch clamping
I checked the expression of ric 3 with WB and this gave a nice result. The channel , alfa 7, was not checked with WB , waiting for maxiprep of the tagged vector.

Any tips or hints are appreciated

wasabi,

What concentration of ACh are you using? If i remember correctly alpha 7 needs to be quite high.