Cell Lysis - (Dec/26/2007 )
I am using BL(21)DE3 cells. I have been getting good expreeion of the protein but it is being retained inside the pellet that is GEL is showing band in the pellet not in the Lysate (supernatant).
I have been using strategy as follows:
1) Inoculate starter LB (5 ml) in the morning with fresh colony.
2) Inoculate Autoinduction media with o.1% inoculum. ( auto-induction media not IPTG induction)
3) Next day in the morning that is around 18 hours culture is used for harvesting
4) spin at 7000 rpm for 20 min
5) wash two times with buffer containg HEPES and MgCl2 pH 7.4
6) Suspend (1gm pellet is suspended in 5 ml of buffer both for washing purposes as well as further procedures) in
the same buffer and add o.2M EDTA+10mg/ml Lysozyme.
7) Incubate at room temp for around 30 min. (mild shaking)
8) Sonicate at 50% for 20 sec pulse with 10 sec off cycle.
9) Spin at 15000 for 15 min.
GEL SDS-PAGE 12.5% at 100 Volts for around 2.5 hours.....
Your protein is in the inclusion body, which is found in the pellet. People use denaturing condition to dissolve proteins then gradually dialyze the denaturing agent away to make it soluble. Do a search for the posts on this subject (inclusion body).
Why you add lysozyme together with EDTA?
generally, EDTA will chelate metal ions and inhibit enzyme activity, I have not seen someone else add EDTA in lysis buffer.
You may check these links for reference: