Immunofluorescence with transfected cells - (Mar/18/2010 )
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!
If the cells that you are using express Myc and you need to detect the transfected Myc, then you definitely need the antibody control! This is so that you can determine the native level of expression, anything above that is probably from your transfection. It is important for any experiment that your controls are the correct one, for IF, antibody controls are very important - you should have both no primary Ab and no secondary Ab controls, the former of which is to determine the level of binding of the secondary only, and the latter to determine the native fluorescence of the tissue/cells.
bob1 on Mar 19 2010, 07:26 AM said:
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
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?