E.Coli Lysis: To add Lysozyme or not to add Lysozyme, that is the question - (Oct/02/2014 )

Hello,

I made an error in my purification today (I have saved the pellets though). I had forgotten to add lysozyme to my ecoli sonication mix. So my samples were diluted up and sonicated and spun down. 

I was curious: Is the lysozyme actually essential for the lysis step when using a probe sonicator? I know it weakens the cell wall of the bacteria and makes it lyse more easily.

Most protocols I can see on the internet have lysozyme added, but some do not, for example. the EMBL website guidance does not feature lysozyme when sonicating:

http://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/sonication/

Lysis buffer

• 50 mM Tris-HCl pH 7.5
• 50-200 mM NaCl*
• 5 mM DTT
• 1 mM PMSF

 

Thanks in advance for your thoughts and comments,

LB

Lysozyme is not necessary if you are sonicating, though it can help in certain situations by making the required sonication times less. The efficacy of lysozyme also depends on the buffer in which you are lysing (e.g. EDTA helps, but that can make it not compatible with traditional NiNTA purification) and how much cell mass you have, etc.

In general though, a single freeze/thaw cycle followed by sonication is sufficient for lysing e.coli.

Thanks for this. Each lab has their own way of doing things, but I ran the protocol anyway without the lysozyme and I managed to recover all my protein! Lysozyme not required!!