I've just started working with Human Aortic Endothelial cells. I'm trying to tag Moesin or L-Asp proteins and over-express them.
Does anyone have a protocol for this?
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#2
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Hmmm, I think it's working
Posted 30 November 2009 - 03:39 PM
Buy/beg/borrow/steal a vector with your tag of choice (GFP, HA, V5, flag...etc.), clone the gene of interest into it, transfect vector containing gene, western/immuno with antibody against tag and/or gene of interest.
#3
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Posted 01 December 2009 - 01:35 AM
For HAECs lentiviral transduction or Amaxa might be the best options, I am not sure if you can use Lipofection with these guys and get a decent efficiency.
#4
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Posted 01 December 2009 - 02:54 AM
bob1, on Nov 30 2009, 11:39 PM, said:
Buy/beg/borrow/steal a vector with your tag of choice (GFP, HA, V5, flag...etc.), clone the gene of interest into it, transfect vector containing gene, western/immuno with antibody against tag and/or gene of interest.
I have a GFP tagged Vector.
However, i have no clue what to do regarding Cloning or Transfection.
I've only started in the lab and im not sure of the protocol used for these.
#5
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Posted 01 December 2009 - 08:13 AM
Hmm.. don't you have a supervisor, labtech, postdoc or other labmembers willing to show you how it's done there?
#6
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Hmmm, I think it's working
Posted 01 December 2009 - 03:31 PM
cm13, on Dec 1 2009, 02:54 AM, said:
bob1, on Nov 30 2009, 11:39 PM, said:
Buy/beg/borrow/steal a vector with your tag of choice (GFP, HA, V5, flag...etc.), clone the gene of interest into it, transfect vector containing gene, western/immuno with antibody against tag and/or gene of interest.
I have a GFP tagged Vector.
However, i have no clue what to do regarding Cloning or Transfection.
I've only started in the lab and im not sure of the protocol used for these.
OK. Good start. Cloning is relatively simple process. I suggest you look at a copy of Sambrook, Fritsch and Maniatis; Molecular cloning: a laboratory manual. It has all the basic protocols and background for cloning. Recent editions might even cover transfection.
Also take note of Warsel's comments, they sound like they have used these cells before, and learning off lab mates is a good thing, they will be able to help you a lot more and quicker than some guys on the internet. Don't be afraid to ask for help, you are wasting your own time re-inventing a process that may well be done routinely in a lab near you.
