Protein localization studies - (Feb/23/2012 )

Hi all,

In order to assay for the localization of a protein within the cell, a protein for which no antibody is available, one can either use GFP or tags, such as HA, FLAG, HIS and myc.

I was wondering what the advantage is, if any, of using GFP? I was told that the large size (230 aa) of GFP frequently interferes with the correct folding of the protein, a problem that is much less relevant with the small tags. So why use GFP at all? Are there notable disadvantages to the tags?

If you decide to go for a tag, for the sole purpose of a fluorescence localization study, how do you decide which tag to go for?

As for GFP and its friends: is there a general rule for deciding whether a C-terminal fusion or an N-terminal fusion should be used? Would you say it is required to try both, if only for the sake of control?

Thanks!

I have definitely found that even small tags can interfere with the function of a protein in the cell. The effect of the tag can be minimised by putting a sufficiently long linker between the tag and the protein, such that the tag is physically separated (though then you have the problem - does the linker interfere?). linkers are usually composed of alanine and arginine residues to minimise the effect of the charges.

Personally, I work on the hypothesis that smaller is better, and so try to use as small a tag as possible.

Tags are certainly valid for testing the function of proteins, so long as you can then further verify that the same function occurs without the tag. Even GFP doesn't stop nuclear shuttling of many proteins, so it doesn't appear to inhibit some functions much, if at all.

gfp allows for fluorescent detection of the protein-gfp complex without any other processing (although you can also detect gfp with antibodies). the other tags require another detection option (eg anti-flag).

I'm reading now a paper in which both a YFP fusion and a FLAG tag were used to localize a protein. The FLAG, however, was used only in a Western blot; only YFP was used in fluoresence microscopy.

I am really wondering what the reasoning for that is. There are anti-FLAG fluorescent antibodies available, here: http://www.cellsignal.com/products/3916.html. Why then perform double work?

The paper is:


Daniel Karcher, Dietrich Koster, Anne Schadach, Anja Klevesath and Ralph Bock (2009). The Chlamydomonas Chloroplast HLP Protein Is Required for Nucleoid Organization and Genome Maintenance. Mol Plant 2:6 (1223-1232).

Perhaps the fluorescence microscopy was performed on live cells?

both C-terminus-tagged and N-terminus-tagged should be tested;

GFP-tagged proteins in cells can be anlayzed by:
confocal/fluorescence microscpoy
FRET analysis
IHC/ICC
IP and IB