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 (S DS)-protein in the presence of cascade blue in agarose gel electrophoresis using barbital buffer was found to be equally feasible in the absence of th e fluorescent marker and using Tris-Tricinate buffer, provided that SDS was loaded with the sample but not contained in the catholyte. That fluorescen t detection is thought to be due to the formation of a moving boundary betw een leading SDS and trailing barbital, or Tricinate buffer. This hypothesis is supported by the following evidence: Ii) The fluorometrically detected band disappears with addition of SDS to the catholyte; Iii) band area is pr oportional to protein and/or SDS load; (iii) mobility of SDS-proteins diffe ring 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 prop erties of a stack comprising moving boundaries of any analytes with net mob ilities intermediate between SDS (or micellar sos) and the trailing buffer constituent at their regulated very high concentrations. The steady-state s tack 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 buffe r 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 mol ecular sieving (open-pore gel and small-sized protein) contributes to the u nderstanding of moving boundaries in gel electrophoresis, but in view of th e narrowly defined conditions, under which this stack forms, is of limited practical significance for the gel electrophoresis of SDS-proteins.