Hi,
I am trying to generate a stable MARC 145 cell line. I successfully transfected the cells with phCMV vector containig the gene of interest, transiently. However, after selecting with G418 for about 1 month, the Immuno fluorescence and WB analysis does not show the expression of the protein.
Any of you have an idea what/where the problem could be?
Please let me know your suggestions.
Thanks
Maali
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science noob
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Posted 02 November 2012 - 04:57 PM
Does your plasmid have a GFP tag on it? Or do you have to quantify expression by WB and immuno?
How was the initial expression of your protein of interest? And what was the transfection efficiency?
Have you done a kill curve with your untransfected MARC cells? you sure that your current concentration kills all cells (=nothing left in the plate)? This is very important as you don't want untransfected cells to recolonise your culture.
How was the initial expression of your protein of interest? And what was the transfection efficiency?
Have you done a kill curve with your untransfected MARC cells? you sure that your current concentration kills all cells (=nothing left in the plate)? This is very important as you don't want untransfected cells to recolonise your culture.
Edited by science noob, 02 November 2012 - 05:00 PM.
