protein tag for ICC/IF - (May/06/2008 )
Hi All~
I'm hoping to get some suggestions on good tags I can use for immunofluorescence on mammalian cells. I'm already using Myc and HA on other constructs and GFP/YFP/CFP as well as dsRed are giving me non-specific effects in my system (empty vector) so I definitely can't use these. Flag doesn't seem to work all that well (I need to see sub-cellular localization) and to be honest, I'm stuck. I was thinking of using beta-galactosidase since I know expression doesn't have these non-specific problems but it looks like cloning may be a bit more difficult than anticipated. Just thought I'd throw it out there and see if anyone has any ideas. Thanks for any suggestions!!!
I'm hoping to get some suggestions on good tags I can use for immunofluorescence on mammalian cells. I'm already using Myc and HA on other constructs and GFP/YFP/CFP as well as dsRed are giving me non-specific effects in my system (empty vector) so I definitely can't use these. Flag doesn't seem to work all that well (I need to see sub-cellular localization) and to be honest, I'm stuck. I was thinking of using beta-galactosidase since I know expression doesn't have these non-specific problems but it looks like cloning may be a bit more difficult than anticipated. Just thought I'd throw it out there and see if anyone has any ideas. Thanks for any suggestions!!!
sometimes, not the tag but the resistance factor of the expression factor (if there is one) may cause trouble;
I wonder why fluorescent protein-tags do not work in your system as they are ideal for subcellular localization in many cases...
Problem is I'm looking at an event in the cell that GFP nonspecifically inhibits. Great for localization, bad for determining if my GOI plays a role in this event. Currently everything I have is GFP-tagged and I need to switch it to a tag that won't inhibit the event and will also be good for assaying localization of the expressed protein.