Fluorescence tag - question asked by BioSaint (Feb/01/2009 )

BioSaint had PMed me this question.

Would people help BioSaint about this question?


hi Minnie,
I have got a question, I guess you're working with FACS often. I need to couple amyloid peptide to fluorescence material without harming its conformation. If you know some molecules in this regards, kindly pass on the information.

Have a nice weekend!

Saint..

Are wanting to tag the endogenous protein or are you talking about expressing a fluorescent labeled protein? I'm not sure what you are wanting to do but if you are wanting to express a tagged protein that you can sort by FACs check out the flash and reash system from Invitrogen. You just add a six amino acid tag to the protein and then use a cell-permiable labelling reagents that are only fluorescent when it binds the tag. They have multiple colors and this is all done in live cells. I've personally never used this system but it looks amazing and has a huge range of applications.

Thanks Minimouse for posting it here, I have the same in the Neuroscience thread too. Thanks rkay for your post.

*** Well, I want to couple some fluorescent tag to 'synthetic' Aß peptide, as you know its hydrophobic and tough to fix the conformation. If someone got some ideas about tagging the fluorescent stuff without harming the conformation of the peptide, it ll be use that for many of my experiments.

Thanks in advance.

I'm a bit confused. You mentioned in the neuroscience thread (which I can't find now) that you want to do this work in live cells but you say in this post that it's tough to fix the conformation. Are you wanting to follow your fluorescently labelled protein in live cells or are you able to fix the cells then use antibody detection?

If live cells, you have a problem since the protein expressed must have the fluorescent tag expressed as well. These are large proteins which often mess with structure, function, localization, ect. There is no way that I know of to use antibody detection in live cells so all you have is the protein expressed inside the cell. This is where I suggest the six amino acid tag that can be labelled with cell-permiable reagents.

If you can fix your cells than you have much more options. Just put a HA or myc tag on the protein and then you can use antibodies after fixation. These are very small tags and I have used them extensively for IF. There are even really good primary antibodies conjugated to a wide range of fluorphores.

Of course, any tag has the potential to mess with your protein but using the smallest is best. You can add a linker between the tag and your protein (especially if you want to try the larger fluorescent proteins themselves) to help get the tag away but this can also just create more problems. You may wind up having to screen multiple tags before finding the one you want. Just because one tag interferes with your protein's conformation doesn't mean that another one won't. You may need to try putting the tag in different positions (CT vs NT).

BioSaint on Feb 3 2009, 11:24 AM said: