1. Grow starter from a single colony on LB medium with
antibiotics O/N.
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.
LYSIS
BUFFER
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.