Tuesday, December 14, 2010
GFP-based quantification of proteins in the cell - don't try that at home!
This is something that come out of our tracking experiments in J. Elf lab. I guess we will never publish any quantitive account of it, so it guess it constitutes perfect blogging material.
GFP and its derivatives are widely used to label and track proteins in live cells. This way people follow localization of the protein of interest and accessing changes its concentration.
And here is where the fun begins. GFP has a fluorofor that is bringt only in a) oxidized b) deprotonized state. Therefore redox potential and pH of the cellular environment have profound effect on the brightness of GFP. Cultural media per se can change GFP behavior dramatically, and this is something outside of the cell...
Another problem with GFP is that it can radically affect stability of the fusion protein. We are not even talking about function here, we are talking about the number of molecules per se.
While struggling with our Dendra2 GFP variant and RelA_Dendra2 fusion in vivo we observed all these problems constantly. Unhappy cells were dark, with GFP in the dark state (pH? redox? you simply don't know!) and the numbers of proteins you detect were invariably much lower than what is estimated for the wt, un-tagged RelA.
But all this still does not stop brave souls from using GFP for single molecule quantification of bacterial proteins en masse. Whole library of YFP (yellow fluorescent protein) fusions was created and their copy number as well as diffusion characteristics were analyzed, and then systems biology happened to the dataset. No one knows what exactly does it all mean and how the numbers of detected YFP-fused proteins relate to numbers of the wt proteins, but this is details.
Bogdanov AM, Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov S, & Lukyanov KA (2009). Cell culture medium affects GFP photostability: a solution. Nature methods, 6 (12), 859-60 PMID: 19935837
Taniguchi Y, Choi PJ, Li GW, Chen H, Babu M, Hearn J, Emili A, & Xie XS (2010). Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science (New York, N.Y.), 329 (5991), 533-8 PMID: 20671182
Brian P. English, Arash Sanamrad, Stoyan Tankov, Vasili Hauryliuk, & Johan Elf (2010). Tracking of individual freely diffusing fluorescent protein molecules in
the bacterial cytoplasm arXiv q-bio arXiv: 1003.2110v1