Wiki/Team:Warsaw/protocols

Purification of His_Z_alpha and His_Z_omega

Culture E. coli producer strain in 3 ml of liquid LB medium + kanamycin for 8 hours. Then use it to inoculate 200 ml of liquid LB medium + kanamycin supplemented with 0,5 mM IPTG and grow it overnight. In the morning spin down the culture (5000 RPM, 10 min, 4 deg. C). Resuspend the pellet in PBS buffer and disrupt cells by sonication. Spin down sonication mixture (13200 RPM, 10 min, 4 deg. C) and discard supernatant – purified protein is present in sonication debris. Resuspend it in sterile ice cold ddH2O and store at 4 deg. C.

Culture, induce and disrupt E. coli in the same way as to purify His_Z_alpha. The protein is present in supernatant (about 10% of total protein) and can be added to selection medium without further purification.

We use "Plasmid Mini" plasmid DNA isolation kit from A&A Biotechnology and follow the protocol of producer.