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

At any time in vitroor in vivoexpressed unlabeled proteins have to be quantified it is difficult to find a reliable method, especially with nonpurified samples. Quantification viaprotein activity can result in too low levels if the proteins analyzed tend to aggregate into inactive forms. Here, wild‐type green fluorescent protein (GFPwt) was expressed in high amounts in vitrousing the Rapid Translation System 500 based on Escherichia colilysates. Fluorescent activity was determined in dependence of oxygen and compared to total protein levels. In the presence of low amounts of oxygen only 16% of the whole GFPwt amounts were detectable viadetermination of fluorescence activity. A reliable method to easily quantify whole protein levels even without specific antibodies and without purification steps by simple sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Coomassie blue staining is described.