In-gel refolding allows fluorescence detection of fully denatured GFPs after SDS-PAGE

Abstract

Green fluorescent proteins (GFPs) have been widely used as fusion tags, especially to visualize subcellular localization and dynamics of the fused partner proteins. Also, GFPs serve as fluorescent tags in size-exclusion chromatography and native-PAGE, facilitating the evaluation of expression levels and quality of the expressed fusion proteins. However, the fluorescent detection of GFPs is generally incompatible with denaturing SDS-polyacrylamide gel electrophoresis (PAGE), where the samples are heat-denatured before loading. Accordingly, detecting GFP-fused proteins after SDS-PAGE usually relies on western blotting with anti-GFP antibodies. To enable in-gel fluorescence detection of SDS-PAGE-separated GFPs, some protocols employ mild denaturing conditions to keep the GFPs intact. However, such mild denaturation sometimes results in partial denaturation of the proteins and irregular electrophoretic mobility that is not proportional to their molecular weights. Here, we demonstrate that the fully denatured GFPs can be refolded within the gel by cyclodextrin-mediated removal of SDS in the presence of 20% methanol, enabling the in-gel fluorescence detection of the GFP-fused proteins. The protocol is compatible with subsequent total protein staining and western blotting. Although future studies are needed to clarify the scope and generality, the technique developed here would provide a simple, time- and cost-effective alternative to the immunodetection of GFPs. HighlightsO_LIFully denatured GFPs could be in-gel refolded to enable fluorescence detection. C_LIO_LI? cyclodextrin effectively removed SDS to assist in the in-gel refolding of GFPs. C_LIO_LIGFPs amenable for in-gel refolding include GFPuv, EGFP, turboGFP, and sfGFP. C_LIO_LICompatible with total protein staining and western blotting. C_LIO_LIThe in-gel refolding procedure was successfully applied to two turboGFP-fusion proteins. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/615947v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@19fe9b7org.highwire.dtl.DTLVardef@1a927a8org.highwire.dtl.DTLVardef@6aa42borg.highwire.dtl.DTLVardef@1400b78_HPS_FORMAT_FIGEXP M_FIG C_FIG