Bio-Rad Fluorescent Protein Stains - Manual (Page 8)

6
2.6 Background Reduction (Optional)
Note: The dye in Flamingo fluorescent gel stain has very low fluorescence when
not protein-bound. A background reduction or destaining step is normally not
required but the following step will slightly lower background staining and may
allow some proteins to be more sensitively detected.
Carefully pour off the stain solution and replace with an equal volume of 0.1% (w/v)
Tween 20. Cover the tray, place on a rocker or shaker and agitate gently for
10 minutes. Rinse gels with distilled or deionized water prior to imaging.
2.7 Gel Imaging
The primary fluorescence excitation maximum of Flamingo fluorescent gel stain is
at 512 nm. There is a minor excitation maximum at 271 nm. The fluorescence
emission maximum is at 535 nm (see Section 1.8 Fluorescence Characteristics).
Flamingo fluorescent gel stain therefore gives the most sensitive results when used
with visible laser light-based imaging systems equipped with 473, 488 or 532 nm
laser light sources. The Molecular Imager FX system is particularly well suited for
imaging gels stained with Flamingo fluorescent gel stain, and provides high-resolu-
tion images suitable for proteomics applications. If the accompanying software
does not have a have a preprogrammed imaging function for Flamingo fluorescent
gel stain, the image settings for SYPRO
® Ruby stain may be used. If an alternative
laser-based scanner is used, choose imaging settings that match the fluorescence
characteristics of Flamingo fluorescent gel stain. Examples of compatible excitation
sources and emission filter combinations include the following:
• Green laser (532 nm) with 555 nm longpass emission filter
• Blue laser (473 nm) with 530 nm emission filter
• Blue laser (488 nm) with 560 nm longpass emission filter
Flamingo fluorescent gel stain may also be used with UV transilluminator-based
systems such as the VersaDoc imaging system. However, the limit of sensitivity
with UV transillumination will not be as low as that achievable with a visible laser
light-based imaging system.
If UV transillumination is used, make sure that the transilluminator has 300 nm
bulbs installed.
10003321A.qxp 8/19/2005 11:50 AM Page 6