I am breaking open my recombinant e.coli cells (20% solution in tris sucrose buffer) by lysozyme treatment. After the treatment, the solution turns very viscous. Then I centrifuge the solution at 1,00,000 g for 1 h. Still the supernatant is viscous and the pellet is hard to dissolve to see the proteins in insoluble fraction on SDS PAGE. Now I would like to try DNAse I treatment but I don't how much amount of the enzyme is required to be added. The EMBL protocol suggests to use it at a concentration of 1ug/mL of the suspension but the enzyme available from vendors is either in units/uL or Kunits.
20k x g for 10-30min should be sufficient to pellet insoluble/membrane fractions, and shearing the lysate through a small gauge needle several times will make the DNA much less viscous (sonication also works). You could also add a few microliters of Benzonase solution, as long as it has Mg and the salt concentration is
20k x g for 10-30min should be sufficient to pellet insoluble/membrane fractions, and shearing the lysate through a small gauge needle several times will make the DNA much less viscous (sonication also works). You could also add a few microliters of Benzonase solution, as long as it has Mg and the salt concentration is
Your buffer indicates that you are trying to isolate protein in the periplasm (for using sucrose), but you want to check solubility? sorry if I am mistaken. By the way, I never used DNAse or lysozyme for cell lysis (or prior to sonication), except when I am in the bioprocess development stage in which I am required to remove DNA contamination and/or must avoid the use of physical/mechanical force to disrupt the cell.
if you want to check for protein solubility, the easiest way to do is by re-suspend your cells in standard buffer (say PBS or TBS) and then sonicate. centrifuge at 12,500 g for about 20-30 minutes. take the supernatant and re-suspend the pellet in the same amount of volume with PBS/TBS. Into each you can add loading buffer with DTT/BME and then run SDS PAGE. Your protein is insoluble if you detect it in the pellet fraction, soluble if it is in the supernatant. Further, if your protein is in the periplasm, the size would be (slightly) smaller due to the missing of signal/leader peptide (which responsible for facilitating transfer from cytoplasm to periplasm). If the size is larger (or the same as that of your protein in the pellet), it is in cytoplasm. When I am sure that the protein is in the periplasm, then I (normally) employ osmotic shock (still not including lysozyme or DNAse, except for the aforementioned reason).
For analytical purposes, I resuspend the cells in buffer, followed by by treating for 10 min at 37 °C with 1 mg/ml lysozyme, and then 5 min at 37 °C with 1 % Triton X-100 (final concentrations). This effects complete lysis of the cells with no need for sonication. To reduce viscosity, I use Benzonase® from Roche at a final concentration of 25 u/ml. I avoid pancreatic DNase I because I'm told that it may cause problems due to protease contamination. Resuspending the mixture of debris + possible inclusion bodies forming the pellet after centrifuging the lysate is indeed difficult because it is very sticky. Sonication is the best way I found to disperse it in order to wash it with fresh buffer and eliminate contamination from the soluble fraction.
Dear Mangal,
In old times, the best conditions for the bacterial DNA hydrolysis after E.coli lysis were next: i) cell - 500 millions - 2 billions, volume 10-50 ml;
ii) low salt and 10 mM MgCl2-5 MM CaCl2;
ii) pH 8.5-9.5;
iii) :DNAse I (RNase free, Wathington) - 5microg/ml,
iv) 15 min - 0-4*C.
Good luck!
@Edward Michelini, thanks for replying, I tried centrifugation at 30,000 g for 30 minutes but the pelleting was not good, that’s why I did it at 1,00,000 g for 1 h which completely made the lysate clear. I liked the idea of using the needle for breaking DNA. I cannot do the sonication as our probe got damaged which will take a while. How much benzonase needs to be used per ml for a 20% solution of the cell pellet?
@Wangsa Tirta Ismaya, actually I am following the general protocol for lysing E.coli cells using gentle, heat induced enzymatic lysis given in the Molecular Cloning A laboratory manual by Green and Sambrook.
@Pierre Béguin, Although I am optimizing it for analytical purpose, I have to do it at preparative scale too. So any other suggestions.
@Yuri F. Drygin, Thanks a lot.
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