Immunofluorescence with transfected cells
Posted 18 March 2010 - 05:29 AM
I have a problem with detecting transfected cells using Immunofluorescence. I transfected the cells with pcDNA3.1(+)-myc vector, which contains my target gene. And I used the empty pcDNA3.1(+)-myc vector as control . Then I used Immunofluorescence to detect the expression of my target gene. When I do Immunofluorescence in control (in order to find a fluorescence intensities suitable for experimental group ) including first anti-myc antibody, there is a high background , I can not detected any fluorescence in experimental group .And when I omited the first anti-myc antibody the result is perfect. I think I should choose to omit the first anti-myc antibody in control group, because the cells of control group ( transfected with empty pcDNA3.1(+)-myc )also expressed the myc. Who can give me some suggestions !
Thanks a lot!
Posted 18 March 2010 - 03:26 PM
Posted 18 March 2010 - 11:07 PM
Thanks very much! The problem is whether I use the cells transfected empty vector (pcDNA3.1-myc) as a negtive control to do the immunofluorescence following the step done in positive transfected cells. However, the empty vector also expresses the myc tag, if I use these cells as a negtive control, it maybe mask the positive signal in positive transfected cells (which express the myc-fusion protein).Yours Tao
Posted 12 December 2011 - 08:44 PM
hope you were able to troubleshoot the problem. I am having a similar problem in the sense that when I perform WB, my empty vector and my vector with the target gene all show a similar single band. Any suggestions are welcome. did you change your myc antibody?