I have to do some screenings with Lactobacillus and Bifs in mammalian cells. I will carry out the study of the cell lysate. I think, the most easy way is sonication. Which power/time/number of pulses would you recommend?
Also, which test would you recommend better to check if the sonication worked? I am thinking protein analysis by BCA protein assay. I have done some trials sonicating for 10 s on ice and 3 pulses. Then, I analysed proteins levels before and after sonication, and sometimes I didn't get a great increase after sonication. Is this normal? Could be that many cells were already lysated before sonication? (they were frozen)
Hi Alaa,
Many thanks for your advise. However, I would prefer dont use lisozyme as I will have to use that lysate on my cells and I prefer some mechanic way rather than chemical way for lysis, so that I dont have to add any chemical component.
Thanks again,
Cristina
I have sonicated E. coli as follow with excellent results. I collect the cells by centrifugation, wash them with 50 mM Tris–HCl buffer (pH 8.0) + 1 mM PMSF (protease inhibitor) and sonicate in ice by ten cycles of 30 second bursts with 30 second cooling intervals (amplitude 90%, cycle 0.5 in a Branson Sonifier 450 W ). The resulting crude extracts are centrifuged at 20 000 rpm for 20 min at 4ºC to remove cell debris. I usually determine protein concentration by using the BCAProtein Assay kit of Thermo Fisher Scientific.
Marzio.L.D. et.al. found 3 min. and 50 seconds necessary, alternating 10 seconds sonification and 10 secnds pause needed for St.thermophilus in PBS sol. at room temp.
Thank you so much for the suggestions!! just few questions more:
- In how many mL you sonicate? I was doing only in 0.5 mL as I want the lysate concentrated as much as I can if possible. Do you think this is too small volumen?
- How much increase in proteins after sonication would you say is good enough to consider that the sonication is working? I only got the double in one sample..the rest were just a bit more....
Thanks a millions in advance!
They tested 5 mL, but send me an e-mail to [email protected] and I will send you the work
Andres gracias por tu respuesta!! muy completa la verdad :)
Is not 10 cycles too much??! Im afraid that sonication could affect to peptides/proteins, estability..I cannot add the protease inhibitors as they interfere with my assay....
For disrupting Anabaena cells I usually use only 5 cycles and comes well. For E. coli I use 10 cycles and the proteins of interest are relased in native state. Maybe you have to use some protein or peptide of your microorganism as control of complete cell disrupting. If you have antiserum for any intracellular soluble protein, you may performe a western blot after sonication using the soluble extract to be sure that the intracellular protein is released. Good luck Cristina!
Thanks Andres!
Im doing screenings with several different strains and we are not looking at an specific protein so that wont work for me. However, I can have a look in the literature for some protein from a couple of strains and do the western blot just to confirm the protocol is working ...
Thanks!!!
Lactobacilli and bifido are really hard to destroy, much more thna E.coli. Did you try some plating after sonication to see viability. The sonication process has to be carried on ice especially for long period of sonication, more over the sonication process is inhibited by high cell concentration. From the answer you gave it seems that Bead beatting would be a far more better approach than sonication.
Hi Frederic,
I didnt try any plating to test the viability as I think that I may get a reduced viability but cells may be still no lysed (just dead). Actually, I checked them under the microscope after the lysis and they seem exactly the same than before lysis (any change in shape, etc.). I have also checked proteins content and there was no differences. So, I should try something else such as bead beatting as you suggest.
Many thanks for your suggestion,
Cristina
Hello Cristina,
How did the lysis went with you. I am carrying a similar work with different strains of lactobacilli, so I was wondering what procedure you used and how did you check for cell lysis?
Many thanks in advance
Muna
I am also interested as i am using lactobacillus and bifido strains. Please share the outcome.
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