Fig. 3:

Optimization of the 2D-TLC. Wild-type cells (strain AJW678) were labeled at 37°C in mMOPS supplemented with 0.8% pyruvate, harvested, processed, and subjected to 2D-TLC. (A) Separation of a lysate that was not precipitated. Note that the [³²P_i] obscures the acetyl-P signal. (B) Separation using the following solvents: first dimension buffer, 0.75 guanidine HCl (19), second dimension buffer, as described in Materials and Methods (14). Notice the streaking in the first dimension that obscures the acetyl-P signal (arrow) and the lack of resolution along the right edge caused by prolonged development in the second dimension. (C) Separation using the McCleary and Stock system (14). Although the signals in the lower left corner are well-resolved, the acetyl-P signal is obscured by streaking in the first dimension (arrow). (D) Separation using the optimized McCleary and Stock solvent system. Note that the acetyl-P signal is well resolved (arrow).