Hello everybody,
I have transfected Hela cells with histagged 45kDa protein in pcDNA3 and selected stable cells with geniticin for 3 weeks. At the end selection, I could not detect any exogenous protein with WB, altough all of negative control cells (no DNA) have died.
What may be the reason? Any suggestions or ideas are appreciated,
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Stable cells survive with antibiotics but no exogenous expression detected with
Started by UmiYasi, Dec 21 2011 01:27 PM
2 replies to this topic
#2
mWright
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Posted 22 December 2011 - 01:14 AM
Hello. I'm no good in chemistry, but maybe you have some acid in those cells?
Or soap? Maybe you dropped something in there?
Just saying.
___________
mkv player
Or soap? Maybe you dropped something in there?
Just saying.
___________
mkv player
#3
UmiYasi
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Posted 24 December 2011 - 02:17 PM
Hi mWright.
I have tried stable transfection for several times, this is not for the fisrt time, so it can not be accidental I guess. I have transfected 4 different mutants and a wild type of my proteins and with all of them I got no exogenous expession at all
I am using Lipo2000 and selecting with 500ug/ml geneticin. After 24 hours of transfection I split my cells into bigger plate, as they become too confluent. But I can clearly observe that -DNA cells are dying but others survive.
Have no idea what is going on....
I have tried stable transfection for several times, this is not for the fisrt time, so it can not be accidental I guess. I have transfected 4 different mutants and a wild type of my proteins and with all of them I got no exogenous expession at all
I am using Lipo2000 and selecting with 500ug/ml geneticin. After 24 hours of transfection I split my cells into bigger plate, as they become too confluent. But I can clearly observe that -DNA cells are dying but others survive.
Have no idea what is going on....
