Stacking of unlabeled sodium dodecyl sulfate-proteins within a fluorimetrically detected moving boundary, electroelution and mass spectrometric identification.
The previously reported fluorimetric detection of sodium dodecyl sulfate (SDS)-protein in the presence of cascade blue in agarose gel electrophoresis using barbital buffer was found to be equally feasible in the absence of the fluorescent marker and using Tris-Tricinate buffer, provided that SDS was loaded with the sample but not contained in the catholyte. That fluorescent detection is thought to be due to the formation of a moving boundary between leading SDS and trailing barbital, or Tricinate buffer. This hypothesis is supported by the following evidence: (i) The fluorometrically detected band disappears with addition of SDS to the catholyte; (ii) band area is proportional to protein and/or SDS load; (iii) mobility of SDS-proteins differing in mass is the same at agarose concentrations up to 3%; (iv) lowering of protein mobility by increase in gel concentration and/or increase in the size of the SDS-protein leads to band disappearance. Fluorescent detection of the band is like to be nonspecific and due to the light scattering properties of a stack comprising moving boundaries of any analytes with net mobilities intermediate between SDS (or micellar SDS) and the trailing buffer constituent at their regulated very high concentrations. The steady-state stack of SDS-proteins in the size range of 14.4-45.0 kDa, and the transient stack of an SDS-protein of 66.2 kDa have lent themselves to electroelution and characterization by mass of the proteins after removal of SDS and buffer exchange using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. The possibility to form a stack of protein between leading SDS and trailing buffer anions under conditions of weak molecular sieving (open-pore gel and small-sized protein) contributes to the understanding of moving boundaries in gel electrophoresis, but in view of the narrowly defined conditions, under which this stack forms, is of limited practical significance for the gel electrophoresis of SDS-proteins.
Yefimov
S
S
Laboratory of Cellular and Molecular Biophysics, NICHD, National Institutes of Health, Bethesda, MD 20892-1580, USA.
Sjomeling
C
C
Yergey
A L
AL
Chrambach
A
A
D002021:Buffers; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods; D011506:Proteins / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D002021:Buffers; D011506:Proteins; D012967:Sodium Dodecyl Sulfate