A highly uniform UV transillumination imaging system for quantitative analysis of nucleic acids and proteins.
The digital fluorescent imaging for documentation and analysis of gel electrophoretic separations of nucleic acids and proteins is widely used in quantitative biology. Most fluorescent stains used in postelectrophoretic analysis of proteins and nucleic acids have significant excitation peaks with UV light (300-365 nm), making midrange UV (UV-B) as the excitation source of choice. However, coupling quantitative CCD imaging with UV is difficult due to lack of uniformity found in typical UV transilluminators. The apparent amount of those macromolecules depends on the position of the gel band on the imaging surface of the transilluminator. Here, we report the development and validation of a highly uniform UV transillumination system. Using a novel high density lighting system containing a single lamp formed into a high density grid, an electronic ballast, a phosphor coating, and a bandpass filter to convert 254 nm light produced to 300-340 nm, uniformity of </=5% CV has been achieved, compared to >80% CV observed in typical UV transilluminators. This system has been used for the quantitative analysis of electrophoretically separated nucleic acids and proteins (CV</=8%) without uniformity correction by software, and continues to outperform typical transilluminators when a uniformity correction is applied.
Chakravarti
Bulbul
B
Keck Graduate Institute of Applied Life Sciences, CA, USA. bulbul_chakravarti@kgi.edu
Louie
Melissa
M
Ratanaprayul
Wongrat
W
Raval
Alpan
A
Gallagher
Sean
S
Chakravarti
Deb N
DN
D000818:Animals; D002417:Cattle; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000494:pharmacology; D009696:Nucleic Acids / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013194:Staining and Labeling; D014172:Transillumination; D014466:Ultraviolet Rays
D005456:Fluorescent Dyes; D009696:Nucleic Acids; D011506:Proteins; D012710:Serum Albumin, Bovine; D004247:DNA