page_title: SDS-PAGE
section_title: Sample preparation
hierarchical_section_title: SDS-PAGE / Procedure / Sample preparation
caption_attribution_description: Disulfide_reduction_by_DTT
context_page_description: SDS-PAGE, is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. The combined use of sodium dodecyl sulfate and polyacrylamide gel allows to eliminate the influence of structure and charge, and proteins are separated solely on the basis of differences in their molecular weight.
context_section_description: During sample preparation, the sample buffer, and thus SDS, is added in excess to the proteins, and the sample is then heated to 95 °C for five minutes, or alternatively 70 °C for ten minutes. Heating disrupts the secondary and tertiary structures of the protein by disrupting hydrogen bonds and stretching the molecules. Optionally, disulfide bridges can be cleaved by reduction. For this purpose, reducing thiols such as β-mercaptoethanol (β-ME, 5% by volume), dithiothreitol (DTT, 10 millimolar) or dithioerythritol (DTE, 10 millimolar) are added to the sample buffer. After cooling to room temperature, each sample is pipetted into its own well in the gel, which was previously immersed in electrophoresis buffer in the electrophoresis apparatus.
  In addition to the samples, a molecular-weight size marker is usually loaded onto the gel. This consists of proteins of known sizes and thereby allows the estimation (with an error of ± 10%) of the sizes of the proteins in the actual samples, which migrate in parallel in different tracks of the gel. The size marker is often pipetted into the first or last pocket of a gel.
caption: Disulfide reduction by DTT
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