Need help on STAT1-GFP nuclear translocation - (Jan/25/2013 )
You will see an impressive change in Stat1 phosphorylation after IFN treatment independent of starvation. IF and western will agree if your reagents are working correctly. Personally, I don't starve.
Typical IF protocol suggests 10min of cold methanol permeabilization. Have you tried 10min instead of 30min of methanol permeabilization? Would 10min be insuffisant?
When there is almost no IFN in serum, starving would not be necessary. But if there is a relative high concentration of IFN in serum, it could be problematic. Recently the quality of the serum in my lab is not so sure...
Looks like you are correct. Ten minutes of -20C methanol is what they recommend now: http://www.cellsignal.com/support/protocols/IF_methanol.html
Good point about potentially having some IFN in the serum. I use heat inactivated serum and we test every new lot of serum prior to purchase, so maybe that's why this isn't a problem for our lab. Based on your images, high basal signaling isn't your problem, though.
I find a website which has a lot of info http://www.copewithcytokines.de/cope.cgi?key=WISH
According to this site, WISH is used to assay IFN alpha and gamma. So it seems that WISH is more sensitive to IFN than its original clone, HELA cell.
I found some WISH cells and hope I would get some good news...
I know this topic has gone cold but i was curious to find out whether Fresna-ni had managed to show translocation of Stat-1 by using the EGFP tagged plasmid from Addgene. I have tried the same approach IFN treating three different cell lines and i couldn't find any translocation taking place using a live cell confocal. I haven't sequenced the plasmid yet but I am curious to know if there is a problem with it..
willc on Mon Feb 3 10:50:51 2014 said:
I know this topic has gone cold but i was curious to find out whether Fresna-ni had managed to show translocation of Stat-1 by using the EGFP tagged plasmid from Addgene. I have tried the same approach IFN treating three different cell lines and i couldn't find any translocation taking place using a live cell confocal. I haven't sequenced the plasmid yet but I am curious to know if there is a problem with it..
You are relatively unlikely to get a response from Fresa-ni, as their last post was in this topic and they were last active on the 17th June last year.