I want to perform an untargeted lc-ms analysis of bacterial metabolite. Generally, we break down the cell of this bacteria by dissolving the pellet with methanol and then give heat treatment for 30 minutes at 55 degrees celsius to extract carotenoid.
But as heat treatment may destroy heat-labile metabolites, that is why I want to go for probe sonication. I have taken stationary phase culture and dissolved the culture in PBS buffer. Then I have done the sonication process (40% amplitude with 30 sec on/off for 20 minutes).
PBS buffer composition (100 ml, 1X PBS, pH- 7.4)
NaCl- 0.8 gm
KCl- 0.02 gm
Na2HPO4- 0.144 gm
KH2PO4- 0.0245 gm
What procedure should I follow to break the cell so that metabolites remain intact?
Hello,
I think you should include detergents like deoxycholate, Tritonx100, SDS in the extraction buffer. And it is better to make the lag period in the sonication step 15 seconds instead of 30 seconds.
I also advise you to have a look at the book " Methods of enzymology" Volume 22. Which deals with how you lyze different types of biological membranes.
Good Luck
@ Hasan
Thank you so much
NaCl- 0.8 gm
KCl- 0.02 gm
Na2HPO4- 0.144 gm
KH2PO4- 0.0245 gm
TritonX-100 - 0.5%
Hi Sanhita Saha ,
As Hathama Razooki Hasan suggested, the addition of detergents like SDS, Tween-20, Triton-X in your lysis solution will be helpful for the degradation of gram-positive bacteria.
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