The detection was performed by adding 100 l of a 1 mg/ml 2, 20-azino-bis(3-ethyl benzthiazoline-6-sulfonic acid) (ABTS) substrate solution (Roche Applied Technology, Mannheim, Germany), and the ELISA readings were recorded at 405 nm after incubation. case of are frequently used to determine the effects of vector elements on certain characteristics, such as recombinant protein build up (rpa) and protein stability. However, transient gene manifestation in tobacco has been reported to result in inhomogeneous protein accumulation. Many different factors are thought to be involved in this observation, such as the availability of phytohormones [9], [10], sugars [11], [12] and phenolic compounds [13], [14]; the presence of cell wall proteins influencing bacterial motion [15], [16]; uneven infiltration [17]; gene silencing [18]C[20]; a general incompatibility between and the flower [21]; and leaf cells senescence [22]C[26]. In transient manifestation assays, which are used to compare different manifestation constructs, factors such as the availability of hormones or sugars affect all transformation events in the same way and are consequently considered to have a only a weak effect on inhomogeneous protein accumulation. As different parts of a leaf or of a single flower are used for those assays, we expected leaf senescence and leaf position to have a stronger impact on inhomogenious protein accumulation than the additional factors listed above. We assessed the influences of the growth stage and leaf position within the rpa levels of three model proteins and found that both leaf age and the position within the leaf have significant effects. To control for such heterogeneous protein build up, infiltrating and consequently extracting the TSP from all the leaves of an entire tobacco flower would be necessary. Using this method, only a single construct could be analyzed per flower, and the testing of multiple constructs would be a time-consuming and laborious process. We hypothesize that a sufficiently exact measurement of the rpa can be obtained via the HS80 analysis of only a few randomly chosen flower parts rather than the analysis of an entire flower. Based on this hypothesis, we developed a high-throughput leaf disc-based infiltration assay that allows for the recognition of manifestation constructs that result HS80 in the highest rpa and protein stability for the efficient production of recombinant proteins. Results and Conversation Analysis of the Inhomogeneity of Recombinant Protein Build up in Transiently Transformed Tobacco Leaves Three different proteins, the reddish fluorescent protein DsRed from (GV3101) transporting the binary manifestation vector pTRA, which contained a gene for one of the three model proteins. After incubation, leaf discs were cut according to the schema demonstrated in Number 1A to assess the effects of leaf age and position within the leaf within the rpa levels. Following extraction, the TSP was identified using the Bradford assay, and the recombinant protein concentration was identified with an ELISA (phl p 1, M12) or a fluorescence measurement (DsRed). The entire process was repeated twice for each model protein. To illustrate the inhomogeneity of the rpa levels, we summarized TIAM1 the results in HS80 the contour plots demonstrated in Number 1BCD. As demonstrated from the graphs, the protein accumulation varied considerably between biological replicates and depending on the leaf age and position within a single leaf. The average ratio between the highest and least expensive accumulation measured for each protein (maximum/min percentage) was approximately 11 (Table 1). Consequently, the construct-based effects within the rpa levels will become masked by background noise until an 11-collapse or higher difference is accomplished. There are several examples in which expression constructs have been optimized to improve the yield of a target protein. These approaches lead to a 1.2C12-fold increase in rpa (Table 2), but because different.