Dear Jaya Aseervatham ,

May be some internal problem with your process, the problem is i thing your protein is not clearly bind in exact receptor, but from your info i have not feel any major problem, in this condition G2/S the cells should need to express,

if possible to try with some other protein tag? or any pre activated tag means you can able to know, my Head have very clear knowledge about this, if you no able to get clear answer for this means i will request him to give response to this problem,

Good Luck & Regards

R.Selva....

Thank you. That would be of great help. Can I request him to help me out with this problem, as Iam new to this work and any help would be appreciate.

If your protein (GT) inserted properly means you will get clear expression.,  do you estimated the protein level in the system before expression?

if possible means as selva chemist suggested, insert one pseudo active protein and check the expression sure you will get.,

Hi,

You can try FACS sorting to sort cells expressing both rfp and gfp. You can also try to co transfect both the rfp and gfp simultaneously and sort cells expressing both rfp and gfp by Facs. It worked for me. I check for fluorescence at a higher magnification 63x by seeding cells on cover glasses.