Getting the most from fluorescent proteins, Part 2

On Feb 3^rd in our previous blog entry on fluorescent proteins, we discussed some basic tips on setting yourself up for success with fluorescent protein based experiments. Here are some more ideas to help boost your imaging success:

1.    Know your background.

All cells contain endogenous fluorescent materials which can confound image interpretation, especially when your fluorescent protein signal is weak.  Make sure you’re familiar with the autofluorescence of your cell type before starting your FP experiments: take some images of non- expressing control cells using the same filters and excitation wavelength as you plan to use for your FP imaging.

Keep in mind that for mammalian cells, autofluorescence is confined mainly to the blue and green regions of the visual spectrum, while in other organisms (e.g. plants, yeast, and bacteria), some cell types may contain fluorescent compounds in other regions of the spectrum. For any given species and cell type, there is likely to be a wavelength “window” with the least autofluorescence; try to choose a fluorescent protein in this wavelength range for maximum signal above background.

2.    Sometimes two (or more) FPs are better than one.

If you are having trouble obtaining sufficient fluorescent signal from a fluorescent protein fusion construct, consider adding an extra copy of the fluorescent protein to boost your brightness.  While this is not recommended unless all else has failed, for low-abundance proteins it can substantially increase the likelihood of detection.  It is possible to create a functional fusion of two or more copies of fluorescent protein in many cases, although the larger size of such a tag increases the chances of mislocalization, so proper controls and validation are essential if you use this technique.  Also, remember that it is generally difficult to use PCR to amplify tandem copies of any gene, including FPs, so restriction-based subcloning is the most reliable way to create multi-copy FP tags.

3.    The best fluorescent proteins don’t stick together!

Truly monomeric fluorescent proteins make the best fusion tags, since they don’t produce localization artifacts due to multimerization. Even weak dimers, such as EGFP and its derivatives, can cause trouble if your fusion protein is at high concentration or in a confined space like a membrane or vesicle.

Are you still using your old EGFP fusion constructs?  If so, make sure to validate your localization results by other methods, or switch to a truly monomeric FP such as mTFP1 or mWasabi.  If you prefer to keep your original constructs, note that any Aequorea GFP-derived FP can be made completely monomeric by adding the A206K mutation.

One final warning — many commercially available FPs that were initially advertised as being monomeric later turned out to be dimers!  With any new FP you try, validate your results before making your conclusions.

Tags: cell marker, cellular, cellular localization, fluorescence, fluorescent protein, FRET, GFP, imaging, mTFP1, mWasabi

Discovery and Development of GFP Rewarded by 2008 Nobel Prize in Chemistry!

Press Release 8 October 2008

The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2008 jointly to Osamu Shimomura, Marine Biological Laboratory (MBL), Woods Hole, MA, USA and Boston University Medical School, MA, USA, Martin Chalfie, Columbia University, New York, NY, USA and Roger Y. Tsien, University of California, San Diego, La Jolla, CA, USA “for the discovery and development of the green fluorescent protein, GFP”.

Glowing proteins – a guiding star for biochemistry

The remarkable brightly glowing green fluorescent protein, GFP, was first observed in the beautiful jellyfish, Aequorea victoria in 1962. Since then, this protein has become one of the most important tools used in contemporary bioscience. With the aid of GFP, researchers have developed ways to watch processes that were previously invisible, such as the development of nerve cells in the brain or how cancer cells spread.

Tens of thousands of different proteins reside in a living organism, controlling important chemical processes in minute detail. If this protein machinery malfunctions, illness and disease often follow. That is why it has been imperative for bioscience to map the role of different proteins in the body.

This year’s Nobel Prize in Chemistry rewards the initial discovery of GFP and a series of important developments which have led to its use as a tagging tool in bioscience. By using DNA technology, researchers can now connect GFP to other interesting, but otherwise invisible, proteins. This glowing marker allows them to watch the movements, positions and interactions of the tagged proteins.

Researchers can also follow the fate of various cells with the help of GFP: nerve cell damage during Alzheimer’s disease or how insulin-producing beta cells are created in the pancreas of a growing embryo. In one spectacular experiment, researchers succeeded in tagging different nerve cells in the brain of a mouse with a kaleidoscope of colours.

The story behind the discovery of GFP is one with the three Nobel Prize Laureates in the leading roles:

Osamu Shimomura first isolated GFP from the jellyfish Aequorea victoria, which drifts with the currents off the west coast of North America. He discovered that this protein glowed bright green under ultraviolet light.
Martin Chalfie demonstrated the value of GFP as a luminous genetic tag for various biological phenomena. In one of his first experiments, he coloured six individual cells in the transparent roundworm Caenorhabditis elegans with the aid of GFP.
Roger Y. Tsien contributed to our general understanding of how GFP fluoresces. He also extended the colour palette beyond green allowing researchers to give various proteins and cells different colours. This enables scientists to follow several different biological processes at the same time.

Read more about this year’s prize

Information for the Public
Scientific Background
In order to read the text you need Acrobat Reader.
Links and Further Reading

Osamu Shimomura, Japanese citizen. Born 1928 in Kyoto, Japan. Ph.D. in organic chemistry 1960 from Nagoya University, Japan. Professor emeritus at Marine Biological Laboratory (MBL), Woods Hole, MA, USA and Boston University Medical School, MA, USA.
www.conncoll.edu/ccacad/zimmer/GFP-ww/shimomura.html
Martin Chalfie, US citizen. Born 1947, grew up in Chicago, IL, USA. Ph.D. in neurobiology 1977 from Harvard University. William R. Kenan, Jr. Professor of Biological Sciences at Columbia University, New York, NY, USA, since 1982.
www.columbia.edu/cu/biology/faculty/chalfie/Chalfie_home/
Roger Y. Tsien, US citizen. Born 1952 in New York, NY, USA. Ph.D. in physiology 1977 from Cambridge University, UK. Professor at University of California, San Diego, La Jolla, CA, USA, since 1989.
www.tsienlab.ucsd.edu
Our congratulations to all of them! We are particularly happy for Roger Tsien, graduate adviser of our own Nathan Shaner and a UCSD colleague and teacher of many of us here at Allele.

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