Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Stable cells survive with antibiotics but no exogenous expression detected with


  • Please log in to reply
2 replies to this topic

#1 UmiYasi

UmiYasi

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 21 December 2011 - 01:27 PM

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,
best

#2 mWright

mWright

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

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

#3 UmiYasi

UmiYasi

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

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....




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.