Subcellular localization of -HA tagged protein - (Jul/20/2008 )
I've tried searching on the methods that one employs to study subcellular localization of proteins, but couldn't come up with much.
I have a -HA tagged protein that I'm inducing using a Tet-On system in CHO cells, and would like to study the localization of the protein. It is hypothesized that the protein is a transcriptional regulator.
What are the methods that I can use to go about studying the localization of this -HA tagged protein?
Thank you very much in advance.
The first thing to try is simple IF. I have used HA for IF and it works very well. Molecular Probes has a really nice Alexa488 primary conjuated anti-HA (mouse monoclonal). I use it all the time on CHO cells expressing my HA construct. This will show you if your protein is primarily in the nucleus. You may want to play with the time of induction as well. Cells that are highly expressing may not be very good at showing the localization since they will have very strong signals and many times once a protein is expressing at such high levels you will see it throughout the entire cell. I find that induction of just 10-12 hours in my Tet-On (using Trex Flp-IN, Invitrogen) is enough to see the expressed protein and get a good look at where it localizes.
Another potential assay you may want to consider is a differential extraction. You permiabilize the cells in a hypertonic buffer BEFORE fixation. This removes proteins not bound to chromatin. As a transcriptional factor/regulator you would expect it to be bound to chromatin at some time, correct? With this assay you could test different stages of the cell cycle, effects of drugs, pretty much any other condition to see if this has an influence on your protein binding chromatin. Check out PMID: 11896191 for the protocol. There are also a couple tricks to doing this with CHO cells I could discuss with you, if you are interested.
Otherwise there are ways to fractionate cells. This way you isolate the cytoplasm from the nucleus and can go on to separate the nucleosolic fraction from the chromatin bound proteins. You run this out on gels and do western blots to show that your expressed protein was in a specific fraction. This is tricky though, especially for overexpressed proteins. It's difficult to not get contaminating proteins in the fractions. You have to do control blots to show that the fractionation was clean. Over-expressed proteins tend to be in all fractions. This is generally a better assay for identifying the localization of the endogenous protein. A paper with a protocol for this is PMID: 10692433.