I am trying to do an enzymatic assay by bacterial cell lysate, but cell lysate i want to prepare without lysozyme treatment. so, suggest me what should i do and how i do?
Hi, if you don't want to use lysozyme, you can try mechanical methods like sonication or chemically use base to lyse. I used the lysis buffer provided in the kit for my E. coli to get plasmid. it's 200 mM NaOH, 1%SDS. lyse for 5 min then neutralise with KAc. It works fine with me but the protocol did state that for harder to lyse strains, lysozyme is suggested.
Not too sure what strain you are using and what downstream application conditions you need, but you can try this =)
Hi there,
Miniprep lysis protocol suggested by Sunny is made for DNA extraction, not protein extraction. 0.2N NaOH and 1%SDS will be denaturing for most proteins and KAc treatment is precipitating a lot of protein material!
You are interested in enzyme activity so you have to avoid denaturing agents. What you could do in a first attempt is to harvest the cells, wash them and resuspend them in a lysis buffer (20mM Tris-HCl pH 7.5, 200mM NaCl, 5mM BME) and sonicate or using French Press to break the cells mechanically. Centrifugation at 13000g will generate a clarified supernantant for enzyme assays (if your enzyme is soluble). If interested in non soluble enzyme you will have to add non denaturing detergent to solubilize the pellet containing membrane and cell débris.
Thank you Mr. Liger as per your concern i will remember to this important step of protein denaturation and enzyme activities. But, i have one query that my bacterial sample is Gram positive bacteria and only lysis buffer will be effective for this as you suggested?
Hi Thanks Dominique for the correction. I didn't realise that. And yes, cos the cells are not only break up by lysis buffer along by also the mechanical stress from sonication or french press
Thank you both of you, now i am doing my work as you suggested and lets see what results i will get...
Dominique Liger Hi! I am also trying to lyse Enterococcus faecalis without lysozyme for intracellular GSH measurement. I just wonder that what can I use instead of BME for lysis buffer. Any suggest?
Hi
Sucrose, mannose or glycerol added lysis buffers for osmolarity change dependent cell lysis is also possible, Besides common physical techniques such as suggested by authors above, without the usage of chemical agents, freeze/thaw cycle induced cell lysis approaches may also be useful for special purposes.
Density gradient centrifugation: rate-zonal or isopycnic could also help to get reduced lysozyme protein concentrates, since discontinuous or step gradients application of sucrose addition possible.
I am not sure these ones convenient for your research, but perhaps they may lead conduce towards new ideas...
Thanks, good luck
Emir...
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