Bacterial Expression

E.coli Expression and Renaturing Protocols - http://www.its.caltech.edu/~bjorker/Protocols/Ben_protocols.pdf E.coli Expression and Renaturing Protocols Bjorkman Lab. 1. High efficiency competent E.coli cells (i) Grow bugs in LB or similar - 500 ml for good batch (ii) When grown to OD 600 of 0.6, spin down gently. (iii) Resuspend in 100mls cold TFB1 and then spin down again (iv) Resuspend in 20mls cold TFB2 - aliquot and store at -80oC. TFB1 - [Read more]

Inclusion Body Protein Isolation Protocol - http://www.its.caltech.edu/~bjorker/Protocols/Isolation%20of%20proteins%20from%20.pdf Isolation of proteins from inclusion bodies Bjorkman Lab. Expression of recombinant proteins Materials: - LB medium - 40% glucose - Amp 100 mg/ml - IPTG 100 mg/ml in PBS Methods - occulate 100 ml LB medium + 1 ml glucose + Amp with 1 fresh colony - [Read more]

PI-PLC Purification - http://www.its.caltech.edu/~bjorker/Protocols/PIPLC_purification.pdf PI-PLC Purification Watson SHEN 8/17/95 1. Inoculate with an overnight culture 1 liter of LB media + 1 X ampicillin. (100ug/ml) 2. Grow at 37 degrees to an OD of approx. 1.0 at 600 nm. 3. Induce by adding IPTG to 1mM. 4. Grow at 30 degrees for 16 hours. 5. Spin down cells in large bottles in GSA rotor: 4,000g x 15 minutes at 4 degrees (5,000 rpm in this rotor). - [Read more]