3 replies to this topic

[mike79]

Hi everyone, I have registered to ask for your help regarding expression and the addition of lysozyme.
My problem is that my desired protein to be expressed is ~14 KD and when I  add lysozyme as part of the expression protocol (about 10mg) I get an inactive protein which I think is just the remaining lysozyme. I use expression by sonication using BL21 cells.
Is there any way  around this? Is using lysoyme necessary for expression and protein extraction, or is there any alternative to lysozyme that is not 14kd?
Thank for your help in advance.

[mike79]

I am really amazed as to how this forum is almost dormant. I was hoping someone might have an input.

[Sheepy]

Hi, you might want to consider a lysis buffer instead of using lysozyme if you are worried about it interfering with your analysis.
I've only used the FastBreak lysis reagent from Promega so that is the only one I can recommend from experience.

[Missle]

Sonication alone can work quite well if you optimize your conditions.  Lysozyme is helpful but not necessary.  If you put your pellet thru a freeze-thaw or two that will increase you lysis as well.  I've also used SoluLyse - a commercial lysis detergent reagent.

Good Luck!