Generation of Transcription Factors in Rabbit Reticulocyte Lysate Depleted of Endogenous DNA-Binding Protein

The rabbit reticulocyte lysate (RRL) is a convenient system for in vitro translation of transcription factors (1 ). By using in vitro-synthesized transcription factors, the time-consuming and often difficult process of protein purification from cellular extracts of in vivo expression systems is avoided. Although the endogenous level of RNA in the RRL is removed by treatment with micrococcus nuclease, there is a substantial level of proteins in the lysate, among them many DNA-binding proteins. These endogenous proteins can interfere with the function of newly synthesized transcription factors. Problems can be caused by signal overlaps in electrophoretic mobility shift assays (EMSA) and by dimerization of endogenous proteins and in vitro-translated products.