Abstract
Sodium dodecyl sulfate electrophoresis (SDS) is a protein separation technique widely used, for example, prior to immunoblotting. Samples are usually prepared in a buffer containing both high concentrations of reducers and high concentrations of SDS. This conjunction renders the samples incompatible with common protein assays. By chelating the SDS, cyclodextrins make the use of simple, dye-based colorimetric assays possible. In this paper, we describe the optimization of the assay, focussing on the cyclodextrin/SDS ratio and the use of commercial assay reagents. The adaptation of the assay to a microplate format and using other detergent-containing conventional extraction buffers is also described.
Highlights
Determination of protein concentration is one of most common assays used in biochemistry
Classical detergent-containing buffers alter the Bradford protein assay in different ways
When concentrated protein extracts are available, which is often the case for studies using western blot techniques, the sensitivity of the protein assay allows the use of minute amounts of detergent-containing samples, which do not induce high background interference
Summary
Determination of protein concentration is one of most common assays used in biochemistry. The most commonly used protein assays belong to two families. In the first family (copper based methods), the biuret reaction is used to detect the presence of peptide bonds. In this reaction, Cu(II) is reduced to Cu(I) by peptide bonds in strongly alkaline conditions. The second family of methods (dye-binding methods) is based on the ability of proteins to bind to certain dyes and to induce an absorption shift in the dyes. The most common method uses Coomassie blue as the dye [3], but metal-dye complexes, such as pyrocatechol-molybdate [4] or pyrogallol red-molybdate [5] have been used for protein assays
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