1. Grow starter from a single colony on LB medium with
2. Dilute the bacterial culture 1:100 into 2xYT medium and grow till OD600=0.6. Induce (with IPTG: 0.4 and 0.8mM). Grow for various time periods (5hrs, ON). Harvest cells by centrifugation (6000 rpm/5min), aspirate supernatant and freeze pellet at -70oC.
3. Resuspend 1.5 ml pellet of bacterial cell culture in 0.75 ml of lysis buffer (see below). Incubate on 30ºC 15 minutes or 30min on ice.
4. Sonicate 3x20’’ till sample is no longer viscous.(Check the sonication procedure)
5. Centrifuge 12,000rpm for 20min at 4oC.
6. Collect sups to new tubes, and re-suspend pellets in 0.75ml lysis buffer.
7. Take 60ml of sup and pellet to new tubes and add 20µl (1/4 volume) 4X sample buffer w/100mM DTT (freeze the rest of the lysates in -20ºC)
8. Boil 5' and load on PAGE-SDS gel.
50mM Tris pH 8.0
10% glycerol (for stabilization of the protein and prevention of aggregation). Glycerol in the protein solution may pose a problem in NMR and structure studies.
0.1% Triton X-100 (for prevention of aggregation of hydrophobic and membrane proteins). Detergents chosen for the lysis solution should be specific to the proteins
100ug/ml lysozyme (its MW is ~15Kd. If studying the induction of a protein of similar MW, you may consider not to add it).
1mM PMSF and /or more anti-proteases (don't add EDTA, if your protein is his-tagged).
DNAse 3U + 2mM MgCl final conc.
Add lysozyme, PMSF right before the experiment.
Add DNAse after sonication.