Subnanomolar detection limit for sodium dodecyl sulfate-capillary gel electrophoresis using a fluorogenic, noncovalent dye.
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.
Department of Chemistry and Biochemistry, Concordia University, Montreal, QC, Canada.
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Harvey
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Bandilla
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Banks
D002021:Buffers; D002826:Chorismate Mutase / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes; D008832:Microchemistry; D008970:Molecular Weight; D011303:Prephenate Dehydrogenase / Q000737:chemistry; D011489:Protein Denaturation; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate
D002021:Buffers; D005456:Fluorescent Dyes; D012967:Sodium Dodecyl Sulfate; D011303:Prephenate Dehydrogenase; D002826:Chorismate Mutase