I'm trying to prove that my protein of interest colocalizes with the centrosome in various ovarian cancer cells (SKOV3, HEY, OVCA). I fused my protein of interest with GFP and did a transient transfection to introduce the construct into each of these cells. I also introduced constructs with only GFP into a separate group of cells as a control. I fixed the cells with 4% PFA and used a pericentrin antibody to confirm any colocalization. The typical GFP signal is seen but, strangely enough, the construct with the GFP alone also tends to develop a very bright structure that colocalizes with the centrosome. As far as I know, this should not be happening. Other research with centrosomes using a similar setup don't show any colocalization between GFP alone and the centrosome. I also see the colocalization with the GFP fusion protein, but it is not as strong as the constructs with the GFP alone. Given the colocalization with the GFP alone construct, I can't really conclude anything regarding colocalization of the centrosome and my protein of interest.
Anyone have any ideas why this could be happening/ how I can stop this from occurring?
Thanks to all.