ABSTRACT
The possibility to quantify free-flow electrophoresis (FFE) data was explored in application to 6 negatively charged polystyrene size standards in the size range of 73 to 762 nm diameter. Peak fraction numbers in FFE were shown to be proportional to mobilities of the particles, determined by capillary zone electrophoresis in the identical buffer. Standard deviations of peak fraction numbers demonstrate a high degree of intra-experimental reproducibility while inter-experimentally, a variability of 1 to 5 peak fraction numbers within 28 fractions was found. A relative mobility (Rf) scale for peak identification in FFE based on the free mobility of the dye, SPADNS, allowed for the utilization of the entire electrophoretic migration path but failed to improve the precision of fraction numbers in view of the substantial zone spreading of the dye. Mobility differences between particles increased upon lowering the ionic strength of the electrophoretic buffer. Peak width increased with particle size in inverse relation to ionic strength.
Subject(s)
Electrophoresis/methods , Microspheres , Polystyrenes , Buffers , Coloring Agents/chemistry , Dextrans/chemistry , Electrophoresis/instrumentation , Electrophoresis, Capillary , Kinetics , Osmolar Concentration , Particle Size , Polymers/chemistry , Reproducibility of ResultsABSTRACT
Five SDS-proteins, ranging in molecular weight from 14 to 66 kDa, were detected without covalent fluorescent labeling by the automated gel electrophoresis apparatus with intermittent fluorescence scanning (HPGE apparatus, LabIntelligence) during electrophoresis in barbiturate buffer in the presence of Cascade Blue. The SDS-proteins were electroeluted from the gel into 220 microl of buffer by a modification of the procedure of Gombocz and Cortez. The electroeluate was freed of SDS, ultrafiltered and subjected to MALDI-TOF mass spectrometry. The masses of the five native proteins were found to be maintained after electrophoresis and electroelution in the presence of the potential contaminants SDS, barbituric acid and Cascade Blue. The procedure of protein transfer from SDS-PAGE into mass spectrometry, without excision of bands, gel maceration and protein recovery by diffusion, therefore is shown to be suitable for the identification by mass of intact proteins derived from gel electrophoretic bands.
