questions about cell lysis - (May/02/2009 )

Hi, there. I met some problems when breaking E. coli (BL21) cells using sonicator. I want to release the expressed enzymes out of the E. coli cell wall to get the crude enzyme lysate. However, I failed due to some unknown reasoms. The probe I used is medium-size type and the maximum power is 120 watts. I usually set to 80-100 % amplitude to break the cell for 3 times, each time around 15 secs. But the lysis buffer containg the cells seemed to be unchanged afterwards. As I known, when the sonication works well to break the cell, the soluton will become quite clear and transparent.

I don't know if anyone here have the experience of sonicating the E.coli cell to release the crude enzyme extract. I really need your help and suggestion. Thanks in advance.

zx0819 on May 2 2009, 07:28 PM said:

Have you tried freeze-thawing in the presence of lysozyme and DNaseI? 2 or 3 rounds gives very good lysis, and is very gentle.

zx0819 on May 2 2009, 10:28 AM said:

Hi,
What is your volume of cells? If its 20-30 mls you can increase the sonication to 6X 30 seconds with 1 minute intervals (but be careful not to over-heat your cells as you might denature your protein). Also lysate may not be clearing if the genomic DNA is not being sheared - I usually treat with DNAse before sonication.

I hate to sound like a salesman when I'm trying to help, but this really is a solution: the Bullet Blender (link). It won't heat your samples like a sonicator will, it's high-throughput, and it'll give you more consistent results. Take a look and see if you think it's a good instrument for your application, and feel free to send me a message if you have questions.

Best of luck!
-Carlton

Carlton H on May 9 2009, 12:59 AM said:

sorry but you do sound like a salesman. Which is acceptable if you are one.



What's wrong with freeze-thaw?

Not sales. Marketing.

Nothing is wrong with freeze-thaw per se, but it'll never be as fast nor as consistent as a good homogenizer (like, *ahem*, the Bullet Blender) Also, if you're going to be doing a lot of lysis and you're aiding the lysis with enzymes, it can get expensive over time.

Cheers,
-Carlton

Carlton H on May 11 2009, 10:14 PM said:

Marketing. Of course. That explains the cool sunglasses...

zx0819 on May 2 2009, 02:28 AM said:

HI

You did not mention your lysis concentration like 10gm cells per lit or 5gm/lit and do you check the Absorbance of the cells before and after lysis.

Nomrally with sonicator you will get 80% cell lysis (small volumes). Check the Absorbance in between each cycle of lysis and determine the lysis percentage. Try not to heat the sample keep in chilling conditions. Viscosity will increase as the cell lysis because of DNA .

Use little concentrated cell slurry for lysis with chilling buffer conditions. you will lyse them easily. Include EDTA 1-5mM , it will enhance the lysis and prevents the proteins from degradation from Mettaloproteases.

Regards
Sudhakar Mutyala