I was trying to make the aqueous stock solution of AHL (3-oxo c12 HSL). I dissolved 3-OXO- C12 HSL (20mg/ml) in ethyl acetate containing 0.01% glacial acetic acid and it was evaporated by passing Nitrogen gas. I got a white precipitate in the bottom of the tube which was insoluble when tried to dissolve in aqueous buffer.
Please suggest what else I can change in the protocol for making the aqueous stock.
Hi , you can see this publication
https://www.researchgate.net/publication/11162037_N-Acylhomoserine_Lactones_Undergo_Lactonolysis_in_a_pH-_Temperature-_and_Acyl_Chain_Length-Dependent_Manner_during_Growth_of_Yersinia_pseudotuberculosis_and_Pseudomonas_aeruginosa
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC128322/
Article N-Acylhomoserine Lactones Undergo Lactonolysis in a pH-, Tem...
Hello Vivek.
My partner worked with the same AHL, and instead of glacial acetic acid, we used 0,05% formic acid with ethyl acetate to prepare our stocks of all AHLs that we use. We had the same situation (white precipitation). Once we prepare the stocks, we put at -20°C, then before of dissolving with the broth, we just resuspend in a vortex. We didn't notice a precipitation in our test, probably because the volume we used was minimal. Still it would be great to know if there is a properly way to dissolve this particular AHL since it is hydrophobic.
I found this, hope it helps...
We find that AHLs are best maintained as stock solutions in
ethyl acetate acidified with glacial acetic acid (1 ml of acetic
acid per 1 L of ethyl acetate). We store them at −20°C in glass
vials. If borosilicate vials are used for this purpose, prior to
adding AHL solutions, glass vials are first treated with 0.2 N
HCl, and then rinsed extensively with deionized water.
Teflon-lined caps should be used for capping vials containing
organic solutions of AHLs. As needed for experiments, serial
dilution of 3-oxo-C12-HSL can be prepared in other organic
solvents (including ethanol, methanol and acetonitrile), however
stability of the compound in these solutions is not known.
3-oxo-C12-HSL is not stable in aqueous solutions where it
converts into tetramic acid, an antibiotic (22). At neutral or
basic pH, AHLs undergo spontaneous lactonolysis (23).
From Chapter 9 "Luminescent Reporters and Their Applications for the Characterization of Signals and Signal-Mimics that Alter LasR-Mediated Quorum Sensing" - Quorum Sensing: Methods and Protocols
http://link.springer.com/protocol/10.1007%2F978-1-60761-971-0_9
Best regards.
Hello Vivek, now I am tryin to find the best protocol to make the aqueous stock solution of AHL (3-oxo c12 HSL), if you found the appropriate protocol, can you share it with me?
Thank you.
Hello Vivek, I would also request you to share the protocol for preparing the stock solution of AHL. Thank you
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