Application of the commercial gel electrophoresis apparatus with intermittent fluorescence scanning to a nonfluorescing protein.
Gel electrophoretic instrumentation has taken a quantum jump forward with the commercial introduction of an apparatus which, after loading of the sample and initiation of electrophoresis, provides real-time gel patterns at desired time intervals, with a computer printout of mobility values characterizing each band and the means to isolate each desired band with known and maximizeable recovery. However, a major limitation of that apparatus has been that it employs fluorescence detection and therefore requires the fluorescent labeling of the macromolecules of interest. That limitation was first overcome by E. Gombocz and E. Cortez (Application Note 8, 1994, LabIntelligence, Belmont, CA) in the detection of nonfluorescing carrier ampholytes. In that application, fluorescent, immobile (uncharged) umbelliferone was added to the gel to provide a uniform background of fluorescence upon excitation at 280-360 nm. The isoelectric carrier ampholyte zones could be detected as inverted peaks due to their reduction of the fluorescence intensity of umbelliferone. A similar approach was applied to a representative SDS-protein, conalbumin-SDS, in the present study, replacing umbelliferone in the gel by a fluorescing paper sheet in contact with the lower external surface of the electrophoresis cell. Passage of the proteins reduced the intensity of the light excitation incident on the fluorescent paper so as to decrease the emitted fluorescence signal and allow for the detection of the proteins as "inverted peaks." Presumably, the reduction of background fluorescence is due to the absorbance at 280 nm of the protein passing through the gel, and reduction of the incident light intensity by that absorbance. The resulting detection of the representative unlabeled SDS-protein by "fluorescence reduction" was found to be less sensitive by a factor of 10-20 than detection of the fluorescently labeled protein (at a molar ratio of fluorescein carboxylate to conalbumin of 1/1). The area of the inverted bands of conalbumin-SDS was found to be independent of migration distance.
Chen
N
N
Section on Macromolecular Analysis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1855, USA.
Chrambach
A
A
D003207:Conalbumin / Q000032:analysis*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005452:Fluoresceins; D005453:Fluorescence; D012680:Sensitivity and Specificity
D005452:Fluoresceins; D003207:Conalbumin