Fluorescein
Fluorescein (ex = 492 nm, em = 525 nm) is one of the most widely used fluorophors and is employed as a detection agent in numerous assays. The Dark Reader is at least twice as sensitive as a UV device for the detection of fluorescein and up to 16-fold when the dye is in a test-tube or in a gel apparatus. (More information on the Walls page.)
Ultra-sensitive Protein Detection
In a recent study, Friedl and Graf (Electrophoresis, 22, 890-895, 2001) compared the detection of biotinylated proteins on nitrocellulose membranes using either:
(A) the fluorescence of fluorescein-labeled streptavidin visualized on a Dark Reader transilluminator, or:
(B) the luminescence generated by the breakdown of luminol (ECL) by peroxidase- streptavidin conjugate.
Method
A simplified summary of the basic protocols for the detection of biotinylated proteins by either (A) fluorescein fluorescence or (B) luminol luminescence is shown in the Figure. In both methods the local biotin concentration was first amplified by the use of the `catalysed reporter deposition` technique in which additional biotinylation is generated in the vicinity of the original biotin. Fluorescence was detected using a Dark Reader transilluminator together with a Hamamatsu CCD camera in Acquire Mode and an exposure time of about 10 sec. Luminescence was detected using Kodak X-ray film and an exposure time of up to 15 min.
ECL Luminescence
Fluorescein plus Dark Reader
fluorescence versus luminescence
Results
The results (Fig. 2, below) show that the Dark Reader-based fluorescence detection technique is much more sensitive than can be achieved using ECL luminescence. With a Dark Reader transilluminator it is possible to detect less than 10 pg of protein, compared with 100 pg plus using ECL. Graf and Friedl also point out several problems with the reproducibility and quantification of ECL signals as well as background problems. These effects can be seen in the wide scatter of ECL data points at lower protein amounts.
Dark Reader and fluoresceina re more sensitive Peak areas of scanned blot profiles plotted as a function of the amount of protein blotted.

The 'fluorescein plus Dark Reader' is at least 10 times more sensitive than the luminescent technique.
Clare Chemical Research

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