Lustig, K.D., Stukenberg, P.T., McGarry, T.J., King, R.W., Cryns, V.L., Mead, P.E., Zon, L.I., Yuan, J. and Kirschner, M.W.
Notes: This methods article describes the in vitro expression cloning technique (IVEC) with detailed methods and materials descriptions. Plasmid or phage cDNA libraries were constructed in appropriate T7, T3 or SP expression vectors, and DH10B E. coli were transformed with the libraries. Plasmid minipreps were performed on pools of bacterial colonies, and the pooled cDNAs were used as templates in TNT® Coupled Reticulocyte Lysate System reactions. Proteins were radioactively labeled, and proteins that were phosphorylated or degraded during mitosis were identified by incubation of radiolabeled library pools with mitotic or interphase Xenopus extracts. Proteins were also tested as putative apoptotic caspase substrates using apoptotic and control Jurkat lymphoma cell extracts. In addition, unlabeled proteins were tested for DNA-binding activity in EMSAs. (2049)