Subnanomolar detection limit for sodium dodecyl sulfate — capillary gel electrophoresis using a fluorogenic, noncovalent dye

Abstract

Picomolar limits of detection are obtained using the noncovalent, fluorogenic dye, Sypro Red. The size separation of four commonly used sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) molecular weight markers with 8% linear polyacrylamide (PAA) as the sieving matrix is used to construct a calibration curve for molecular weight determinations. SDS-CGE purity and molecular weight determination of purified chorismate mutase-prephenate dehydrogenase (CMPD) from Escherichia coli is shown to be comparable in accuracy with slab gel SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A migration time precision study indicates excellent reproducibility. Sypro red labeling of SDS-bovine serum albumin (SDS-BSA) complexes at nanomolar protein concentrations suggests assay detection limits surpassing those of silver staining. This detectability exceeds that achieved in previous SDS-CGE laser-induced fluorescence studies. This approach is expected to be easily adapted for use with commercial polymer formulations and automated instrumentation.

Electrokinetic supercharging preconcentration and microchip gel electrophoretic separation of sodium dodecyl sulfate‐protein complexes

“…Thus, the SDS-protein complexes can be separated according to their size differences. High concentrations of SDS would make proteins separation incomplete or affect fluorescent labeling [25][26][27]. The SDS concentration also affects efficiency of preconcentration by EKS and should be optimized, since the current passing in the electrokinetic injection process is shared between the samples and unbound SDS.…”

Microfluidic single-cell analysis of intracellular compounds

“…Thus, for low-copy-number proteins, concentration limits below 100 nM have to be accomplished. Again, CE-LIF is a suitable method, as detection limits for proteins in the picomolar to nanomolar range can be accomplished for both unlabelled proteins with UV-LIF (Lee & Yeung 1992;Tseng & Chang 2000) and labelled proteins employing LIF in the visible range (Harvey et al 1998;Lee et al 1998). Therefore, it was only obvious that this method was also applied for the single-cell analysis of proteins on microfluidic platforms.…”

LIF detection of peptides and proteins in CE

“…SYPRO Red is a dye originally developed for protein staining after SDS-PAGE. Using 8% linear polyacrylamide containing 0.05% SDS as sieving matrix, the LOD values for standard proteins labelled with SYPRO Red were also in the pmol level [173]. Chiu et al [174] have more recently proposed a segmental filling method in CE for the concentration and separation of milk proteins complexed with SDS and labelled with SYPRO Red, yielding an LOD of 0.10 nM for casein in cow milk.…”