mTFP1 is an excellent FRET donor

Because of its excitation and emission wavelength, sharp excitation and emission peaks, high quantum yield, and exceptional photostability, mTFP1 has always been considered a very good Forster resonance energy transfer (FRET) donor (1). More recently, several groups have investigated the use of mTFP1 in various FRET experiments and imaging modalities and have shown that mTFP1 is indeed one of the best choices (2, 3, 4).

In one recent publication, Padilla-Parra et al (2) tested a number of different FRET couples to determine which was the best for fluorescence lifetime imaging (FLIM)-FRET experiments, and found that the mTFP1-EYFP pair was by far the best pair for FLIM-FRET. This group also confirmed that the fluorescence lifetime decay of mTFP1 fits well to a single exponential, and that the time constant for this decay is unaffected by photobleaching, making mTFP1 an excellent choice for any kind of fluorescence lifetime imaging applications, including FLIM-FRET. This group also notes that it is likely that the use of Venus or mCitrine variants in place of EYFP would improve the performance of this FRET pair even further.

In a mathematical analysis of the potential FRET efficiency of mTFP1 with Venus YFP, Day et al. (3) showed that compared with Cerulean (currently the brightest cyan Aequorea GFP variant), one can expect up to 17% better FRET efficiency using mTFP1. This group went on to characterize the mTFP1-Venus pair in live-cell FRET and FLIM-FRET experiments and showed that it worked as predicted in both cases. They also note that mTFP1 has superior brightness and photostability when compared to Cerulean in live cells, which is consistent with all in vitro data reported previously (1). In a related paper, Sun et al. (4) demonstrated that mTFP1 is also an excellent FRET donor for the orange fluorescent protein mKO2.

Together, these recent independent studies confirm that mTFP1 among the best options when choosing a fluorescent protein as a FRET donor. With its proven track record of successful fusions, mTFP1 is also an excellent all-around performer that will enhance almost any live-cell imaging experiment.

(1) Ai et al., (2006) Biochem. J. 400:531-540.
(2) Padilla-Parra et al., (2009) Biophys J. 97(8):2368-76.
(3) Day et al., (2008) J Biomed Opt. 13(3):031203.
(4) Sun et al., (2009) J Biomed Opt. 14(5):054009.

AlleleBlog Admin, by Nathan Shaner

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Tags: cell imaging, CFP, FLIM-FRET, Fluorescent proteins, FP, FRET, FRET acceptor, FRET donor, FRET pair, GFP, GFP fusion, mTFP1, mWasabi, subcellular localization

Why Allele?

Allele provides you with tools that you will find very helpful.  The two main motives for Allele developed products are:
1) To incorporate the most advanced technologies in the field
2) To provide equal utility as other companies’ equivalent products at a much more reasonable cost.

How did we do it?        By developing technologies internally, in most cases with government grant funding, by in-licensing others’ discoveries, and by listening to you, our customers.

What else do we do?      Conduct basic curiosity-driven research just like most of our customers. It helps to stay on the edge and connect to the community.

Tags: Allele, Allele Biotech, biomedical research, cost effectiveness, cost efficiency, DNA, Fluorescent proteins, iPS, low cost, Protein, reagents, RNAi, viruses

Getting the most from fluorescent proteins

Fluorescent proteins (FPs) are an indispensable component of the biology toolbox, providing a robust and straightforward method to optically label nearly any protein of interest.

While most FPs can be used for a wide variety of experimental setups and conditions, getting the best quality data from your hard efforts requires some forethought. Here are a few tips to get the most out of FP imaging:

1.Reduce pre-measurement photobleaching.

All FPs photobleach upon exposure to excitation light. Some, like commonly used YFPs, bleach rather quickly, while others, such as Allele’s mTFP1, are substantially more photostable. However, even the most photostable FPs can be susceptible to excessive pre-measurement bleaching if precautions are not taken.

While searching for your favorite cell on the microscope, try to use the lowest possible excitation light intensity. Close the shutter when you’re setting up software or other experimental apparatus, and use short exposure times whenever possible during focusing.

2.Consider pH and other variables.

Most FPs are somewhat sensitive to acidic pH. Some, such as mTFP1 and many of the red FPs, are reasonably resistant to pH changes, while others, such as EYFP, are highly sensitive. If you’re imaging acidic compartments such as lysosomes or plant vacuoles, you’re unlikely to see any fluorescent signal if you’re using EYFP or any other pH-sensitive FP, so choose wisely!

Information on the fluorescence pKa (the pH at which 50% of the fluorescence emission is quenched) of new fluorescent proteins is generally easy to find, so do your homework!

3.Be careful with fusions and linkers.

One big advantage of using FPs is that they may be genetically fused to virtually any protein of interest. While FPs usually have a negligible effect on the properties of their fusion partners, it’s always a good idea to double-check and validate data on new proteins.

If you don’t know where your protein should localize, check both N- and C-terminal FP fusions to be sure they give the same results. If not, validate your localization by other methods, such as antibody staining. If you can devise a functional assay for your FP-labeled protein, this is also a good way to be sure the fusion isn’t causing trouble.

If you’re having problems with a particular FP fusion, try a few different linkers between the FP and the protein of interest. Floppy linkers, such as poly-(Gly-Gly-Ser-Gly-Gly-Thr) frequently work well, but occasionally rigid linkers (such as poly-proline) or other sequences will give better results. Unfortunately, the process of optimizing a fusion construct is largely empirical.If you can put in the effort early in your experiments to produce the best possible FP fusion, you’ll benefit greatly in later experiments!

Tags: cell imaging, cellular localization, cellular marker, fluorescence microscopy, fluorescent microscopy, Fluorescent proteins, FPs, FRET