Reliable quantification of in vitro synthesized green fluorescent protein:Comparison of fluorescence activity and total protein levels

At any time in vitro or in vivo expressed unlabeled proteins have to be qua ntified it is difficult to find a reliable method, especially with nonpurif ied samples. Quantification via protein activity can result in too low leve ls if the proteins analyzed tend to aggregate into inactive forms. Here, wi ld-type green fluorescent protein (GFPwt) was expressed in high amounts in vitro using the Rapid Translation System 500 based on Escherichia coil lysa tes. Fluorescent activity was determined in dependence of oxygen and compar ed to total protein levels. In the presence of low amounts of oxygen only 1 6% of the whole GFPwt amounts were detectable via determination of fluoresc ence activity. A reliable method to easily quantify whole protein levels ev en without specific antibodies and without purification steps by simple sod ium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Cooma ssie blue staining is described.