Toxojulius wrote:Thanks for the post. I would like to stay away from proprietary lysis buffers since nobody is allowed to know the exact composition, even though they work well.
I am confused why you don't have ANY detergent in your lysis buffer? I always operated under the assumption that lysozyme needed access to the cell wall. I thought that no access would occur unless there was some detergent to remove the outer membrane lipids of E. coli.
I thought Triton-X would an appropriate detergent to solubilize the cytoplasmic membrane, but I included EDTA in my lysis buffer to chelate the divalent cation crosslinks stabilizing the outer membrane, which should allow access for the lysozyme to attack the peptidoglycan layer. This seemed reasonable to me, but again, the resuspended cells never significantly clarify after incubation in this lysis buffer. I always assumed lysis would be indicated by a decrease in turbidity and an increase viscosity due to released nucleic acid.
I would appreciate some feedback on the composition of my lysis buffer and any advice on lysis of E. coli expressing recombinant protein without the use of sonication.
I am attempting to express a recombinant protein in E. coli BL21(DE3) from a pET vector. I am searching for advice on making a good lysis buffer. I would like to stay away from sonication because it's time consuming, variable, and heats up my sample. The lysis buffer recipe I have now is the following:
10mM Tris-Cl pH 8.0
1mM EDTA pH 8.0
1mg/mL chicken egg white lysozyme (Sigma)
1% Triton X-100
1X Protease Inhibitor Cocktail (Sigma)
Here are my questions:
1. Should I incubate my cells first in the presence of lysozyme only (e.g. 30min on ice), then add the lysis buffer above but made without lysozyme? Is there an advantage of pre-treating cells in lysozyme before adding lysis buffer?
2. Usually I add the above lysis buffer above (with lysozyme) to my cells and incubate them shaking gently at 4C for 30min. then at room temperature for 30min. However, the suspension of cells never significantly clarifies. Is this normal? Are my cells still lysing?
3. When I centrifuge the lysate into soluble and insoluble fractions, the insoluble material forms a pellet that is almost as large as the original bacterial cell pellet. This had me thinking that I wasn't getting good lysis of my cells and I was just spinning whole cells down again. However, after I take off the supernatant and attempt to solubilize this insoluble pellet in 1X SDS Sample Buffer (w/5% beta-ME), the pellet is very, very difficult to solubilize. It takes numerous heating-then-vortexing cycles to get the pellet to go into solution into SDS Sample Buffer. Is this normal? Why is the insoluble pellet so hard to solubilize in SDS? Is there something in my lysis buffer that is causing the pellet to be really difficult to dissolve in SDS Sample Buffer?
4. Does anybody have any suggestion for a good lysis buffer recipe? If so, could you tell me what is in it and why?
Users browsing this forum: Alexa [Bot], Google [Bot] and 1 guest