The band areas of proteins determined by fluorescent scanning in the commercial automated gel electrophoresis apparatus.
An automated gel electrophoresis apparatus, recently available commercially, allows one to follow the band during electrophoresis in real time, and lends itself therefore to an evaluation of bandwidth as a function of migration time (the dispersion coefficient), resolution and band shape. These determinations assume the constancy of band area with migration time and at various gel concentrations. The purpose of the present study was to verify these assumptions. Representative proteins and sodium dodecyl sulfate (SDS)-proteins, either natively fluorescent or fluorescein carboxylate labeled, were found to exhibit band areas which approach constancy as a function of migration time in both agarose and polyacrylamide gel electrophoresis, provided that (i) the protein concentration under the band was low enough to obviate self-quenching of fluorescence; (ii) the separation of the protein of interest from contaminants had progressed sufficiently during the time at which band areas were measured; (iii) the baseline under the peak was sufficiently well defined. However, band areas decrease with increasing gel concentration. Protein peaks exhibited leading and trailing tails. The ratio of the combined tail area to total area appeared to be near-constant at varying migration times. However, that ratio increases with increasing gel concentration. The tail area does not appear to be an artifact of fluorometric detection since it is reproduced upon fluorimetric analysis of the protein eluted from gel slices after electrophoresis. However, it may be due to photochemical destruction under the conditions of repetitive fluorometric peak detection.
Section on Macromolecular Analysis, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
S F
SF
Zakharov
S H
SH
Kwok
H
H
Sokoloff
H T
HT
Chang
S P
SP
Radko
A
A
Chrambach
D000327:Adsorption; D001331:Automation; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation* / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation* / Q000379:methods; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D005782:Gels; D006801:Humans; D011506:Proteins / Q000032:analysis*; D013997:Time Factors
D005452:Fluoresceins; D005456:Fluorescent Dyes; D005782:Gels; D011506:Proteins; C097499:fluorescein carboxylate