Enhanced detection sensitivity of “fluorescence reduction” by shifting the analyte absorbance spectrum and use of a fluorescent paper with higher signal/noise ratio
✍ Scribed by Elena Yarmola; Nong Chen; David Yi; Dr. Andreas Chrambach
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 557 KB
- Volume
- 19
- Category
- Article
- ISSN
- 0173-0835
No coin nor oath required. For personal study only.
✦ Synopsis
Enhanced detection sensitivity of "fluorescence reduction" by shifting the analyte absorbance spectrum and use of a fluorescent paper with higher signalhoise ratio
Nonfluorescing protein bands can be detected by the fluorescence optics of the commercial gel electrophoresis apparatus with automated scanning of the migration path (HPGE-1000, LabIntelligence, Belmont CA), taking advantage of the decrease of emission from a fluorescent paper placed below the gel by the absorbance of proteins ("fluorescence reduction"). That decrease of fluorescence gives rise to an inverted protein peak. Nonfluorescent colorless proteins appear to reduce the intensity of light emitted from the fluorescent paper due to absorbance of incident and emitted light. When the absorbance spectrum only slightly overlaps with the excitation and emission spectra of the fluorescent paper, that reduction is weak, and detection sensitivity in that application is consequently only 1/30 of that of fluorescent proteins. By contrast, when the protein is colored so that its absorbance spectrum overlaps widely with the excitation and emission spectra of the fluorescent paper, the sensitivity of "fluorescence reduction" equals 1/4 to 1/5 of that obtained for fluorescent proteins. Bands detected by "fluorescence reduction" provide a quantitative measure of protein load and mobility. The area of the inverted bands is proportional to protein loads up to 16 pg/lane of the gel tray. A theory of "fluorescence reduction" is presented which accounts for the existence of a linear relationship between band area and load.