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293T cell transfection troubles - Lipofecamine 2000

transfection lipofectamine 293T overexpression

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#1 lissencephalic

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Posted 03 May 2014 - 10:11 AM

Hi!

I've generated a plasmid containing my protein of interest (let's call him Bob) with Gateway Cloning. I've confirmed the insert with Sanger sequencing (primer walking). Bob has only has one exon and is a relatively short transmembrane protein (515AA). The plasmid is approximately 9kb with the insert, and was not linearized for the transient transfection.

I want to do a transfection to overexpress protein Bob in 293T cells. My main purpose is to show that my antibody for Bob works.

So I did the tranfection, followed up by Western Blotting. Good news, my antibody works pretty well (tested 293T untransfected, transfected 293 (two different clones), and psirpg transfected cells (transfection control).
Bad news? Transfected cells show no overexpression.

I've repeated this transfection: I tried 6 well plates and 12 well plates (all reagents scaled, of course). The result is the same.
psirpg transfected cells always show GFP-fluorescence, which I assumed was a good sign for my transfection.

I've plated cells in appropriate numbers, followed the lipofectamine 2000 protocol, harvested protein and run Western.

So what could be going on here? I'm about to plan a new transfection where I will add a plasmid with a NDRG1-insert as a transfection/overexpression control (my group has had good experience with this in the past).
Should I be trying for stable transfection instead of transient - and is this even possible with only Amp resistance?

I'm asking because I want to optimize my transfection. My group has not done a lot of transfection lately, so we might have missed something. Personally I have little experience with cell culturing - but my cells seem healthy, are split whenever they grow to 80-90% confluence, and media is changed appropriately.

edit: end goal is to establish that we have a well working antibody for protein Bob for further functional studies.
 



#2 bob1

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Posted 03 May 2014 - 03:15 PM

What do you mean by overexpression?  - is the target protein expressed by the cells you used and you just wanted to express more by transfection?  If so, how do you know that your antibody is working?  If not and the cells only expressed protein when you transfected - then this is overexpression, and will tell you if your antibody is specific.

 

Note that the best way to determine specificity of antibodies is to do a peptide competition where you take a large excess (10-50x) of the protein/peptide against which the antibody was raised and pre-incubate some antibody with it, before adding to a western blot membrane (run parallel one for no peptide) - lack of the band tells you that there was competition and if the band was specific.



#3 lissencephalic

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Posted 03 May 2014 - 04:28 PM

I'm sorry, yes, I mean "overexpression" as in "transfected cells will express more of target protein".

 

The target protein is expressed by the 293T cells. I have clear bands of the right size in my Western, which is why I assume the antibody is working.

 

Thank you for the tip, I should certainly look into that. :)



#4 bob1

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Posted 04 May 2014 - 01:35 AM

Overexpression is a difficult thing to determine - it could well be that the amount of protein that you are loading on your membrane is saturating for the antibody - so you won't see any increase, no matter how much more protein you add.







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