Quantitative FRET microscopy

There’s a nice set of posts on the confocal listserv today about quantitative FRET microscopy using CFP and YFP.  A good cautionary introduction for those of you thinking about doing FRET microscopy.

As an aside, if you’re interested in biological light microscopy, you’d do well to join the confocal listserv – it covers a lot more than just confocal microscopy and has many knowledgeable and helpful readers.

New Addgene Plasmids

The Addgene newsletter turns out to be a surprisingly good source for news on new fluorescent reporters and optogenetic constructs.  In the March newsletter, there are plasmids for Loren Looger’s new glutamate reporter, iGluSnFr [1], a set of lentiviral vectors for RGB marking, which is like Brainbow for clonal analysis [2], and a new optogenetic channel [3]. December’s newsletter has the calcium reporter GCaMP6, a new split GFP, the Clover and mRuby2 FRET pair, and plasmids for RNA tagging using MS2 and a related system.

This seems like it’s worth keeping an eye on.

References

Fluorogen Activating Proteins

In the past decade, some interesting fluorescent protein dye complexes have been published. These are antibodies which bind a non-fluorescent dye (a fluorogen) and stabilize it in a conformation that makes it fluorescent – for example [1][2]. I’ve been curious about these for a while but the lack of commercial availability made them inconvenient to work with. It turns out that the scFvs from [2] – they called them fluorogen activating proteins or FAPs  – are now commercially available from SpectraGenetics.  There are two versions, a green (FITC-like) FAP (which is presumably the thiazole orange binding scFv) and a far red (Cy5-like) FAP (which is presumable the malachite green binding FAP). The far red FAP comes with two different fluorogens – one which is cell permeable and one which is cell impermeant (the only green fluorogen is impermeant). This means you can distinguish between protein on the cell surface and protein that is inside the cell [3][4].

There are probably other clever things you can do with these; if you try them out, let me know.

References

  1. A. Simeonov, M. Matsushita, E.A. Juban, E.H. Thompson, T.Z. Hoffman, A.E. Beuscher, M.J. Taylor, P. Wirsching, W. Rettig, J.K. McCusker, R.C. Stevens, D.P. Millar, P.G. Schultz, R.A. Lerner, and K.D. Janda, "Blue-fluorescent antibodies.", Science (New York, N.Y.), 2000. http://www.ncbi.nlm.nih.gov/pubmed/11030644
  2. C. Szent-Gyorgyi, B.F. Schmidt, B.A. Schmidt, Y. Creeger, G.W. Fisher, K.L. Zakel, S. Adler, J.A.J. Fitzpatrick, C.A. Woolford, Q. Yan, K.V. Vasilev, P.B. Berget, M.P. Bruchez, J.W. Jarvik, and A. Waggoner, "Fluorogen-activating single-chain antibodies for imaging cell surface proteins.", Nature biotechnology, 2007. http://www.ncbi.nlm.nih.gov/pubmed/18157118
  3. G.W. Fisher, S.A. Adler, M.H. Fuhrman, A.S. Waggoner, M.P. Bruchez, and J.W. Jarvik, "Detection and quantification of beta2AR internalization in living cells using FAP-based biosensor technology.", Journal of biomolecular screening, 2010. http://www.ncbi.nlm.nih.gov/pubmed/20488980
  4. J.P. Holleran, M.L. Glover, K.W. Peters, C.A. Bertrand, S.C. Watkins, J.W. Jarvik, and R.A. Frizzell, "Pharmacological rescue of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) detected by use of a novel fluorescence platform.", Molecular medicine (Cambridge, Mass.), 2012. http://www.ncbi.nlm.nih.gov/pubmed/22396015

ABRF report, Day 2

The highlight of day two of ABRF, for me, was the talk by Robert Campbell on fluorescent reporters. He started off by saying that existing fluorescent proteins were quite good and unlikely to get dramatically better. He spent the rest of his talk discussing his labs work to improve fluorescent protein reporters, where there is a lot of work still to be done.  One of his guiding principles is “redder is better”.

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Direct fluorescent monitoring of RNA levels

The same user who was looking for destabilized fluorescent proteins was also looking for ways to directly monitor RNA levels fluorescently. It turns out that two nice reviews on this subject have recently been published: [1] and [2]. Traditionally, this has been done by using RNA binding proteins that bind to a specific RNA sequence, as in the MS2 system. More recently, however, an RNA aptamer has been selected that binds an analog of the GFP chromophore. The chromophore is non-fluorescent in water, but becomes brightly fluorescent on binding to the aptamer [3]. The chromophore, DFHBI, is now commercially available, which means that this system should be pretty easy to use. I’ve not seen anyone use it yet to monitor RNA levels in cells, but I’ll be curious to see if it takes off. Interestingly, this is not the first fluorogenic RNA aptamer synthesized, but none of the others seem to have been that successful.

References