title: Quantitation of yeast total proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for uniform loading.
abstract: Proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer are difficult to quantitate due to SDS and reducing agents being in the buffer. Although acetone precipitation has long been used to clean up proteins from detergents and salts, previous studies showed that protein recovery from acetone precipitation varies from 50 to 100% depending on the samples tested. Here, this article shows that acetone precipitates proteins highly efficiently from SDS-PAGE sample buffer and that quantitative recovery is achieved in 5 min at room temperature. Moreover, precipitated proteins are resolubilized with urea/guanidine, rather than with SDS. Thus, the resolubilized samples are readily quantifiable with Bradford reagent without using SDS-compatible assays.
authors.affiliation: Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada. Electronic address: hyukhos@yorku.ca.
authors.forename: Hyukho
authors.initials: H
authors.lastname: Sheen
mesh_terms: D002021:Buffers; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate; D013501:Surface-Active Agents
chemical_list: D002021:Buffers; D005656:Fungal Proteins; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate
keywords: Acetone precipitation; Laemmli buffer; Protein precipitation; Protein quantitation; SDS sample buffer; Uniform loading