The goal is to try different denaturing methods for bl21 E.coli however, finding detailed paper on this is challenging as most articles do not seem to inclide the deatils of their denaturing procedure. I am currrently using lysis buffer and sonication but i would like to try something else, any detailed suggestions with reason would be of help. Thank you.
Dear Pumla Manyatsi
what do you mean for denatuering BL21 E.coli?
Do you mean cell lysis for extraction of soluble protein (in non denatured conditions?) or do you mean cell solubilization with denaturation of all proteins for extraction of insoluble and membrane proteins?
As Adam B Shapiro wrote you? Which is your aim?
best regards
Manuele
I actually mean disruption methods, the best methods for disruption other than using lysis buffer and sonication?
For large amounts of cells (e.g. 1 liter of culture grown to OD about 0.5-1), the French press is a good method of cell breakage, if the instrument is available.
Adam B Shapiro, Thank you and what buffer solutions can you recommend other than lysis buffer?
It can also be useful to add DNAseI to reduce the viscosity of the lysed cells as well as protease inhibitors if you find that you experience degradation as well. As you lyse larger samples the amount of genomic DNA present can become extremely difficult to deal with ; the DNAse will help considerably with this.
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