- Ultracentrifugation is only needed if HSLs/HSs should be
quantified in intact cells. A detailed description is given by
[12]. For the quantification of excreted molecules in culture
supernatants, a centrifugation at 15,000gfor 30 min is
sufficient. - Samples should be stored no longer than a month depending
on sample type and acyl-HSL concentration. For details
see [10]. - The incubation time varies depending on the assay. The darkest
wells should be about the color of a transparent blue 1 ml
pipette tip (equals approximately to an OD 450 of 1.0). It is
critical not to incubate for too long, as then the color signal will
be in saturation and the standard curve will be flawed. - No washing step should be carried out before adding the stop
solution. If you are handling more than one plate, make sure
that the incubation time—from adding the substrate until
stopping the reaction—is the same for every individual plate.
Usually it is sufficient to always keep the same order in handling
the plates. - Make sure not to forget any dilution of the samples you might
have applied. For example, the hydrolysis of the samples in
Subheading3.2,step 5, results in a dilution factor of 1.25. In
highly concentrated samples, or samples with problematic
matrix effect, dilution of the samples with PBS to up to 10%
of the original concentration might overcome these difficulties. - You may also use a centrifugal vacuum concentrator at
30–35C. - Settings for MS detection: dry gas flow 10 l/min, dry gas
temperature 200C, nebulizer gas flow 2.0 bar, capillary volt-
age 4500 V, end plate offset500 V, ion energy 3.0 eV, and
collision energy 8.0 eV. The MS was first calibrated on a
reference standard including five masses within a mass range
of 100–1600 m/z.
References
- Chen X, Kremmer E, Gouzy MF, Clausen E,
Starke M, Wollner K et al (2010) Development
and characterization of rat monoclonal antibo-
dies forN-acylated homoserine lactones. Anal
Bioanal Chem 398:2655–2667 - Shaw PD, Ping G, Daly SL, Cha C, Cronan
JEJ, Rinehart KL et al (1997) Detecting
and characterizing N-acyl-homoserine
lactone signal molecules by thin-layer chroma-
tography. Proc Natl Acad Sci U S A
94:6036–6041
3. Cataldi TRI, Bianco G, Frommberger M,
Schmitt-Kopplin P (2004) Direct analysis of
selectedN-acyl-L-homoserine lactones by gas
chromatography/mass spectrometry. Rapid
Commun Mass Spectrom 18:1341–1344
4. Rani S, Kumar A, Malik AK (2011) Occurrence
ofN-acyl homoserine lactones in extracts of
bacterial strain ofPseudomonas aeruginosaand
in sputum sample evaluated by gas
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Detection of Bacterial Quorum Sensing Molecules with ELISA and UHPLC-MS 71