|
 |
|
|
| The optical performance of Dark Reader technology is perhaps most spectacular when viewing Green Fluorescent Proteins. The new generation of red-shifted GFPs have excitation and emission properties that are very well suited for viewing with Dark Reader devices. For example, EGFP (ex/em = 488/507 nm) can be detected, by eye, down to concentrations of less than 100 pM. ECFP, EYFP and DsRed are also highly fluorescent under Dark Reader light. |
|
|
|
The diagram to the left shows the fluorescence excitation spectrum of EGFP superimposed on the light output from a Dark Reader and and 300 nm UV device.
There is an excellent match between the Dark Reader and the EGFP - resulting in bright fluorescence. The output from the UV device though, results in very feeble fluorescence.
|
|
 |
|
|
GFP in Plants
|
|
|
The Dark Reader Hand Lamp is proving very popular for the detection of GFP expression in plants. Dr. Anton Callaway at North Carolina State University provided the accompanying photograph of Nicotiana benthamiana plants inoculated with turnip vein-clearing tobamovirus engineered to express an endoplasmic reticulum-localized form of EGFP.
|
|
|
|
|
|
The photograph was taken 6 days post-inoculation using a DR Hand Lamp. The green fluorescent spots (against a background of red chlorophyll fluorescence) show the expanding foci of virus-infected cells.
Also, a group at a leading agricultural biotech company reports successfully using the Dark Reader Hand Lamp to detect EGFP transgene expression in Aridopsis thaliana.
|
|
 |
|
|
X-Ray Vision !?
|
|
| 1: GFP in Tubes |
|
|
|
|
|
 |
The benefit of using Dark Reader light rather than UV light for viewing GFPs is particularly pronounced when viewing GFPs contained within a glass or plastic unit such as a gel apparatus, Petri dish or test-tube. |
|
| A serial dilution of EGFP (ClonTech) was viewed on either a DR45 transilluminator or a 312 nm UV device. Using the DR45, it was possible to detect, by eye, EGFP down to 50 pM. With UV illumination, the detection limit was over 2 orders of magnitude worse. |
|
|
| 2: GFP in Gels |
|
|
|
|
Using a DR Hand Lamp, red-shifted GFPs can even be viewed as they migrate through polyacrylamide gels. The native gel on the left was loaded with several cell extracts expressing EGFP and actually photographed during electrophoresis. (Courtesy of Drs. J. MacMananman and V. Shellman, UCHSC) A recent report (Madin et al., PNAS, 97, 559-564, 2000) describes the use of the Dark Reader to quantitate GFP in native gels. |
|
 |
|
|
Simple, Safe Selection
|
|
| As illustrated below, distinguishing those bacterial colonies expressing GFP from other colonies is a simple exercise using the Dark Reader. In addition, the absence of harmful radiation from DR units can be crucial to the success of an experiment when viewing in vivo systems expressing GFP. |
|
 |
|
Two E. coli cultures (one expressing EYFP and the other not) were streaked on an agar plate and grown overnight before viewing on either a DR45 transilluminator or a 312 nm UV device.
The GFP- expressing colonies (+) are very clearly distinguishable from the non-expressors (-) when using the Dark Reader. |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
