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The quality of the collected experimental data very much depends on the quality of the biological starting material. Especially the proteome analysis of a highly dynamic system like the germinating and tube-growing pollen grain needs several precautions which allow an accurate and acceptable interpretation of the obtained results. Optimized protocols for pollen collection, storage, and in vitro culture as well as pollen organelle separations are described which help to obtain well-defined and reproducible experimental conditions for the subsequent proteomic analysis.