Fluorescent proteins

Choosing the Right Fluorescent Protein

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!

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Thursday, August 9th, 2012 Fluorescent proteins 1 Comment

Our New Website is Complete, 10% off this week

We recently launched our new website, and to celebrate the launch, we’ll be offering a discount of 10 % on all our products and services from July 18th to July 22nd! Come explore a wide variety of products that Allele Biotech has to offer from viral expression to fluorescent proteins. Examples of services offered include custom lentiviral, retroviral, and baculoviral packaging along with cell production and cell line development. This is our effort to enhance your online shopping experience through our improved shopping cart system. Our mission remains the same; to increase accessibility to innovative molecular biology research tools by offering cutting edge products at a reasonable cost. Please visit http://www.allelebiotech.com and use the code NEWSITE to redeem the offer. We thank you for your support!
For more details click here

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Cell Cycle Assays-Part I

This is the first part of a series of blogs about using fluorescent proteins in cell based assays with established examples, a common theme here at the AlleleBlog.

FUCCI Cell Cycle Sensor

The FUCCI Cell Cycle Sensor is composed of a red (RFP) and a green (GFP) fluorescent protein fused to different regulators of the cell cycle: cdt1 and geminin.

During the cell cycle, these two proteins are ubiquitinated at different time points by specific ubiquitin E3 ligases, which tag them for degradation in the proteasome. The E3 ligases’ activities are regulated temporally and result in the biphasic cycling of GERMINI and CDT1 levels during the cell cycle. In the G1 phase of the cell cycle, GERMINI is degraded; therefore, only CDT1 tagged with RFP is present and appears as red fluorescence within the nuclei. In the S, G2, and M phases, CDT1 is degraded; only GERMINI tagged with GFP is present, resulting in cells with green fluorescent nuclei.

During the G1/S transition, when CDT1 levels are decreasing and GERMINI levels increasing, both proteins are present, so are the tagged fluorescent proteins. When the green and red images are overlaid, nuclei fluoresce yellow. This dynamic color change, from red-to-yellow-to-green, represents the entire cell cycle. This representation can be used to study the effects of elements that may influence cell cycles.

Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H, Imamura T, Ogawa M, Masai H, Miyawaki A.Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008 Feb 8;132(3):487-98.

CCNB1-CyclinB(NT)-GFP

In late S phage, CCNB1 promoter will be switched on to drive the expression of Cyclin B N-terminus-GFP expression; thereafter the fluorescent signal will be switched off at the destruction box in Cyclin B N-terminus at the end of Mitosis phase. During the intervening phase the fusion reporter protein will translocate from cytoplasm to nucleus by the cytoplasmic retention signal in the Cyclin B N-terminus.

Thomas N. Lighting the circle of life: fluorescent sensors for covert surveillance of the cell cycle. Cell Cycle. 2003 Nov-Dec;2(6):545-9.

GFP-PCNA/YFP-PCNA
GFP-PCNA, a fusion of GFP and PCNA, has been widely used as a convenient tool to monitor the progress of S phase. At the onset of S phase, GFP-PCNA translocates into the nucleus; at mitosis the nuclear envelope breaks down and the nuclear accumulation of PCNA-GFP dissipates.

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lenti-shRNA expression vector with Puromycin-2A-RFP for custom RNAi virus packaging only. email: RNAi@allelebiotech.com

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Promotion of the week: prepackaged lentivirus expressing IL15, IL2, $25 off, this week only.

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Wednesday, October 27th, 2010 Fluorescent proteins No Comments

Delivery of RNAi or Cre by Ultrasound-Guided Injection of High Titer Lentiviral Vectors

By Jiwu Wang

According to the Skin Cancer Foundation, skin cancer is the most common type of cancer in the US. Although the skin might seem to be an easy target for gene therapy or RNAi mediated functional corrections, the outer keratinized epithelial cells forms a formidable barrier to delivery of genetic material. The epidermis undergoes rapid turnover, a fact that further complicates gene therapy because gene transfer to skin stem cells would be required for sustained effects.

Before skin gene therapy can be discussed with any practical meaning, a physiologically relevant in vivo model for studying gene function in the context of tumorigenesis and epithelial biology must be established. Studies of gene functions in skin homeostasis in mouse models were mostly performed by labor-intensive knockout methods. Recently, at least two publications have shown that by using ultrasound-guided injection of lentiviruses into amniotic fluids, transgene or shRNA can be efficiently and specifically delivered to epidermis, including skin stem cells, creating a very attractive model for functional studies and therapeutic tests.

Localized injection of high titer lentiviral vectors has been widely used for studying genes in brain development and a few other areas. Instead of injection into animal tissues, Endo et al. injected tiny volume (nl) of high titer lentivirus (10e10 TU/ml) into amniotic cavities within a defined window of embryogenesis [1]. By following fluorescent protein markers (CFP, GFP, YFP, RFP), both Endo et al. and researchers from Elaine Fuchs group demonstrated high efficiency and specificity of delivery to epithelial cells, commonly resulting in multiple genomic insertions of the viral genome.

RNAi against alfa1-catenin was used by Beronja and colleagues as an example to show that loss-of-function analysis can be done rather easily using shRNA/FP bearing lentivirus [2]. nlCre was also delivered to embryos with loxP-flanked transgenes vs wildtype for conditional knockout studies. These new findings should open doors to various experiments and therapies concerning the health of the skin.

1. Endo, M., P.W. Zoltick, W.H. Peranteau, A. Radu, N. Muvarak, M. Ito, Z. Yang, G. Cotsarelis, and A.W. Flake, Efficient in vivo targeting of epidermal stem cells by early gestational intraamniotic injection of lentiviral vector driven by the keratin 5 promoter. Mol Ther, 2008. 16(1): p. 131-7.
2. Beronja, S., G. Livshits, S. Williams, and E. Fuchs, Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos. Nat Med. 16(7): p. 821-7.

    New Product of the Week

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Wednesday, August 4th, 2010 Viruses and cells No Comments

Allele’s pallet of the super star fluorescent proteins

“Photoblog”–just some fun pictures from our notebooks.

    The brightest cyan, green fluorescent proteins, and the brightest ever FP in LanYFP!
The brightest cyan, green fluorescent proteins, and the brightest ever FP in LanYFP!

Ain't they pretty?

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.
2-3 times brighter than EGFP, no cytotoxicity detected

The mWasabi is stimulating

The brightest green fluorescent protein with excellent photostability, carried on 10e8 TU/ml high titer lentivirus.

    The LanFPs express well in bacteria.
Reminding you of icecream

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.
Fluorescence while running in denaturing gel

Can you see the FP bands in the SDS PAGE?

    FPs in SDS PAGE–a closer look
while the gel is still running

Can you see them now?

    FPs in gel cassette over UV lights
Easier to see now than during gel running

Invincible FPs

    FPs in gel cassette under blue LED
The red FP is harder to see because of the black background

Fluorescence in SDS page under blue LED

The purified FPs can be used as “real time” protein markers.

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Wednesday, July 28th, 2010 Allele Mail Bag, Fluorescent proteins No Comments