Green-light transilluminator for the detection without photodamage of proteins and DNA labeled with different fluorescent dyes.
The excitation spectra of Nile red and SYPRO red, two currently used dyes for the fluorescent staining of protein bands in sodium dodecyl sulfate (SDS)-polyacrylamide gels, show an excitation peak in the UV region and another in the visible region (maximum at about 550 nm). Ethidium bromide and other intercalating dyes, e.g. propidium iodide, ethidium dimers, and benzoxazolium-4-quinolinium dimer-3 (YOYO), used for the fluorescent staining of DNA bands in agarose gels also show an excitation peak in the same region of the visible spectrum. We have designed and constructed a green-light transilluminator with an emission maximum at 542 nm. This visible transilluminator allows the detection of protein bands stained with Nile red and SYPRO red with the same sensitivity obtained with a 300 nm UV transilluminator. The green-light transilluminator also allows the detection of about 2 ng of DNA per band in gels stained with ethidium bromide and the other intercalating dyes indicated above. In contrast to the UV transilluminators, the green-light transilluminator does not produce photodamage of DNA even after long exposures (10 min). This makes this transilluminator very useful for preparative work. Furthermore, the green-light transilluminator does not require UV safety equipment and, consequently, it can be very convenient for teaching laboratories.
Alba
F J
FJ
Department de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Bermúdez
A
A
Daban
J R
JR
D000818:Animals; D002417:Cattle; D004247:DNA / Q000032:analysis*; D004278:DNA, Superhelical / Q000032:analysis; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods*; D008027:Light; D010078:Oxazines; D011506:Proteins / Q000032:analysis*; D014172:Transillumination
D004278:DNA, Superhelical; D005456:Fluorescent Dyes; D010078:Oxazines; D011506:Proteins; D004247:DNA; C044808:nile red