All my proteins were expressed in the E.coli, and each one only 0.5 g. How to destroy the cells for following protien isolations?
The CelLytic B 10X from Sigma was tested. However, about 50% cells were not destroyed. Which steps were wrong? The freeze/thraw cycles should be carried before or after adding the cell lysis, which is composed with CelLytic B 10X.
DEar Huan Qi
Wihch volume of Cellytic B did you use?
I never used it but i used the Cellytic express (C1990) and it worked quite well.
Normally i'm using 10ml of the Cellytic solution 1X for 0,5g of cells.After cell resuspension, the cells were incubated 30' at RT in gentle shaking and then centrifuged at high speed to recover the soluble fraction.
You can find more information about it at the following link on my blog;
https://proteocool.blogspot.com/2021/08/tips-of-week11-chemicalenzymatic-cell.html
In my experience the main limit of Cellytic is the costs. Sonication is a possible nice alternative that on the basis of the sonication tip that you are using can work well from very small biomass as 0,1g to larger biomass as 10-20g.
Generally with sonication about 10ml of lysis buffer (eg Tris20mM, NaCl300mM, imidazole 10mM pH8 in case your first purification step is the IMAC) work quite well.
Sonication cicle need to be optimized to guarantee cell distruption but not increase the temperature of the sample and the cicles and the pauses beetween the cicles can change on the basis of the sonicator power, probe diameter and sonication volume but since the cicle is optimized it work quite well. Sonication need to be performed in ICE and the sample temperature.
an example of sonication in small volume is available at:
https://proteocool.blogspot.com/2021/05/tip-of-week-6-rapid-protein-expression.html
good luck
Manuele
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