Analysis of bovine serum albumin unfolding in the absence and presence of ATP by SYPRO Orange staining of agarose native gel electrophoresis

An attempt was made to specifically stain unfolded proteins on agarose native gels. SYPRO Orange is routinely used to detect unfolded protein in differential scanning fluorimetry, which is based on the enhanced fluorescence intensity upon binding to the unfolded protein. We demonstrated that this dye barely bound to the native proteins, resulting in no or faint staining of the native bands, but bound to and stained the unfolded proteins, on agarose native gels. Using bovine serum albumin (BSA), it was shown that staining did not depend on whether BSA was thermally unfolded in the presence of SYPRO Orange or stained after electrophoresis. On the contrary, SYPRO Orange dye stained protein bands in the presence of sodium dodecylsulfate (SDS) due to incorporation of the dye into SDS micelles that bound to the unfolded proteins. This staining resulted in detection of new, intermediately unfolded structure of BSA during thermal unfolding. Such intermediate structure occurred at higher temperature in the presence of ATP.

Automated summary

This study attempted to stain unfolded proteins specifically on agarose native gels. It was found that SYPRO Orange dye barely bound to native proteins but effectively stained unfolded proteins. The dye also stained protein bands in the presence of SDS, indicating the presence of an intermediate unfolded structure.