I will be expressing three soluble proteins in E. coli BL-21-T1 cells (Sigma competent cells). The plasmid has a T5 promoter and is induced by IPTG. Many investigators site that the lysis step is just about the most critical step in isolating heterologous proteins. I will lyse the cells via freeze thaw, sonication as well as lysozyme treatment (all done on ice). Is it absolutely necessary to add DNAse and RNAse to the lysis buffer? I am getting some conflicting advice on this.
Thanks for any advice!
Well if you are subjecting your cells to proper sonication then you really don't need to add the DNase/RNase to your lysis buffer. Sonication is a pretty harsh method of breaking open the cell and cellular contents. DNA and RNA should go through mechanical shearing during sonication. In fact, sonication is the most common method of mechanically shearing DNA when required. Addition of protease inhibitors on the other hand would be recommended, esp during sonication and most definitely if you expect the protein to be in the soluble fraction. We usually use the Roche Complete tablets for this in our lab. In addition we also usually add PMSF (serine protease inhibitor) at a final concentration of 2mM.
If you are sure that the protein is in the soluble fraction of the lysate in my opinion, using BL21 cells, you can directly centrifuge the lysate (16.000 rpm for 30 min at 4°C) without DNase/RNase or sonication treatments and isolate the supernatant. Otherwise the sonication could be enough to increase the yield of the purification without the addiction of DNAase/RNAase.
Hi Christopher,
I never use RNase in protein purification protocol and do not see a reason to do it. I would recommend to use DNase if you lyse your cells by sonication, because it helps to shorten the sonication time, which is more healthy for protein. Alternatively you can use French Press or Multiflex for lysis, then you definitely do not need DNase.
Hello there!
The more you avoid adding reagents to your protein purification, the better.
You should compare samples treated with sonication and sonication/DNase. Run an agarose gel with both samples (and an untreated sample!) and verify if just sonication is enough to remove DNA in your case.
Good luck!
No need to add Dnase or Rnase , just cell lysis by sonication (or freezing thawing) then centrifuge are enough. your protein will be in the supernatant.
Hie Gaughan! in my opinion its not necessary to add DNASe and RNSe during the procedure of yeast cell the yeast cell lysis..we regularly lyse the yeast cells in our lab..you better use the french press for the yeast cell lysis though its cell wall is quite rigid....you can use some additional things like freeze thaw your cells then go for frenceh press or else you can add suitable quantity of lyticase and then go for french press for yeast cell lysis.(its not enough to use sonication for yeast cell lysis)..and most of the the time no need to add the DNASe & RNASe...best of luck.
The process of dispersing, disrupting, or inactivating biological materials, such as viruses, by use of sound-wave energy is called sonication. During sonication, DNA samples are subjected to hydrodynamic shearing by exposure to brief periods of sonic waves. adequate shearing can be achieved by increasing the pulse periods or cycles. yoiu dont need to add any DNase/RNase..but if you are worried about sample getting degraded due to prolonged cycles...reduce the cycles in sonicator and add DNase.
You can use a microfluidizer...18000 psi and is the preferred method in our place
I recomend you to add DNAse, RNAase addition is not necessary
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