In 1994 the green fluorescent protein cloned from Aequorea victoria became the first in a long line of genetically encoded labels. Since that time, the fluorescent protein palette has expanded to cover the entire visual spectrum. With so many color variations and options, which fluorescent protein (FP) is best for your research? Three key factors are among the most important to consider: brightness, photostability, and aggregation.
Brightness is the most obvious factor that most researchers consider when choosing an FP. In general, the brighter the FP, the better it will perform under almost all experimental conditions. When evaluating an FP’s brightness, make sure to look at the critical optical parameters — extinction coefficient and quantum yield. The product of these two values for different FPs can be used to directly compare their brightness. Brighter FPs will have lower detection limits (i.e. the concentration at which the FP becomes visible above autofluorescence of other cell components), and will allow imaging with lower excitation light intensity, minimizing the possibility of phototoxic effects.
Photostability has increasingly become a consideration when researchers choose fluorescent proteins. Many FPs, even if they are initially quite bright, will photobleach under continuous excitation during imaging. In order to perform long-term imaging experiments or to do quantitative analysis, an FP with high photostability should be the first choice. Unfortunately, methods for measuring and reporting photostability vary widely in the scientific literature, so be sure to understand how your FP’s photostability was measured before trying to make comparisons!
Aggregation (or oligomerization) has been one of the major issues tackled in the development of FPs. Many wild-type FPs form tetramers, which aggregate badly when expressed as fusion tags in cells. Engineered monomeric forms of many FPs are now available, and these monomeric FPs should always be used when making fusion constructs. For simple expression markers, however, oligomerization is not usually a major concern, and the brightest possible FP should be used in this case.
As with other research tools, doing your homework and reading the primary literature is always the best approach to choosing the right FP for your project!
“Photoblog”–just some fun pictures from our notebooks.
- The brightest cyan, green fluorescent proteins, and the brightest ever FP in LanYFP!
These fluorescent proteins are representatives of the growing family or high quality, new generation FPs engineered to enable experiment previously deemed impossible.
- Cells infected with lentivirus carrying mWasabi. Lentivirus carrying LanYFP will make most cells much more brighter than this.
The brightest green fluorescent protein with excellent photostability, carried on 10e8 TU/ml high titer lentivirus.
- The LanFPs express well in bacteria.
Project planning is under way to test the cytotoxicity of lanFPs in different mammalian cell lines and in vivo with a focus on neurons.
- The FPs fold so strongly that they fluorescence even in SDS-PAGE.
- FPs in SDS PAGE–a closer look
- FPs in gel cassette over UV lights
- FPs in gel cassette under blue LED
The purified FPs can be used as “real time” protein markers.
New Product of the Week 07/26/10-08/01/10: pCHAC-mWasabi-C for expressing mWasabi fusion through retroviral vectors.
Promotion of the Week 07/26/10-08/01/10: Get 3′ TAMRA & BHQ oligo mods for $45 ea & 3′ Dabcyl mod for $20 50 nmol syn scale only/while supplies last- use dbtkrm0726
Allele Biotech has just made a news announcement indicating that researchers from Dr. Campbell’s lab at the University of Alberta, Canada, and scientists at Allele Biotech Drs. Nathan Shaner and Jiwu Wang published a paper in the Journal of Molecular Biology on July 5th introducing a new photoconvertible fluorescent protein mClavGR.
The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and overcome the limitations imposed by the oligomeric structure of the natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 (ICAM-1) both when the membrane is in contact with a T lymphocyte expressing leukocyte function-associated antigen 1 (LFA-1) and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein sub-populations and subsequent tracking of dynamic changes in localization in living cells.
Compared with previously available photoconvertible FPs, mClavGR2 has much improved photostability of the red state under confocal illumination conditions, 3644 over mEOS2’s 2700 and Dendra2’s 2420. Most notable among other advantages of mClavGR2 is its monomeric structure, its highly optimized and relatively rapid folding efficiency, and its high photoconversion effi ciency due to the high pKa of the green state. Its brightness in both the green and the red states is similar to the popular mCherry.
In regard to monomeric state, the monomeric variant of EosFP, known as mEos, was created through the introduction of two point mutations that disrupted the protein-protein interfaces of the tetrameric species. Expression of mEos at temperatures of greater than 30 °C is problematic, but an effectively monomeric tandem dimer variant does express well at 37 °C. mEos2 has been reported to retain some propensity for dimer formation.
We anticipate that this new addition to the toolbox of engineered FPs will be of great utility in imaging of fast protein dynamics in live cells. Experiments to determine whether the advantages of mClavGR2 translate to improved performance in super-resolution imaging applications have been initiated.
Hiofan Hoi(a), Nathan C. Shaner(b), Michael W. Davidson(c), Christopher W. Cairo(a), d, Jiwu Wang(b) and Robert E. Campbell(a)
a University of Alberta, Department of Chemistry, Edmonton, Alberta, Canada T6G 2G2
b Allele Biotechnology, 9924 Mesa Rim Road, San Diego, California 92121
c National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, 1800 E. Paul Dirac Dr., Tallahassee, Florida 32310
d Alberta Ingenuity Centre for Carbohydrate Science
Received 20 February 2010; revised 15 June 2010; accepted 25 June 2010. Available online 5 July 2010.
New Product of the Week 070510-071110: mClavGR greeen-to-red photoconvertible fluorescent protein, catalogue number to be created
Promotion of the Week 070510-071110: Purified lanYFP, bright even in SDS-PAGE gel WITHOUT dye or excitation, great for in gel marker.
LanYFP, identified from lancelet (also known as amphioxus, e.g. Branchiostoma floridae), has been found to have the following properties:
Quantum yield 0.95
Extinction coefficient 150,000
Salt insensitive 0-500mM NaCl
LanYFP has a brightness of 143! For comparison, the brightness of the previously known brightest FPs is 95 for tdTomato, and 34 for commonly used EGFP.
Allele already has been exclusively providing the brightest cyan FP in mTFP1 (brightness of 54); and the brightest green FP in mWasabi (brightness of 56). The confirmation of LanYFP as the brightest ever FP is a major milestone of Allele’s research and development efforts in the fluorescent protein field. We are currently monomerizing LanYFP and another lancelet protein, LanRFP. Once completed, the new proteins should definitely be the FPs of choice for in vivo imaging and FRET with unprecedented utilities.
Promotion of the week 062010-061610: Validated Rex1 Promoter Reporter Lentiviral Particles-1 Vial for $149.00 (ABP-SC-RREX2R1). Save $59 if place an order this week! http://www.allelebiotech.com/shopcart/index.php?c=200&sc=34
New product of the week, recombinant mTFP1, mWasabi, LanYFP, LanRFP, $159 for 125 ug, compare price for 100ug vs 125ug in other companies’ offers, you will know that you are getting a good deal from Allele.
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!
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