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checking transient transfection efficiency


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#1 labrat612

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Posted 23 March 2010 - 06:44 AM

I'm looking for some input:

I need to find a method that can be used to calculate transfection efficiency following my transient transfection. The plasmid I'm using does not contain a GFP site (sad) nor a site that will allow me to do a luciferase type assay, and because I'm doing this in suspension cells, doing a b-gal assay is also proving to be tough (unless someone knows of an alternative protocol that does not involve fixation).

Any thoughts?

Thanks!

Labrat612

#2 bob1

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Posted 23 March 2010 - 03:48 PM

How about a smear on a slide and then some IF for your protein of interest? Otherwise, do a parallel transfection with a GFP or other fluorescent plasmid and assume that the % transfected is similar.

#3 labrat612

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Posted 24 March 2010 - 07:45 AM

[quote name='bob1' date='Mar 23 2010, 07:48 PM' post='63735']
How about a smear on a slide and then some IF for your protein of interest? Otherwise, do a parallel transfection with a GFP or other fluorescent plasmid and assume that the % transfected is similar.
[


I'm not sure what you mean by "some IF for your protein of interest". I had thought of doing say a co-transfection with a commercially-available GFP plasmid, except that I do not have a fluorescent microscope, currently. So I'm trying to do this assay using just a regular light microscope.

I do appreciate the thoughts... do you happen to have any more to deal with this problem?

#4 bob1

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Posted 24 March 2010 - 04:27 PM

IF = immunofluorescence, it would be a direct measure of what proportion of your cells are transfected rather than looking for a GFP. You could otherwise do immunocytochemistry, which is the same as IF, just with coloured (e.g. DAB) rather than fluorescent labels of the antibody.




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