Quorum Sensing

3.4 HPLC Assay

3.4.1 Column Wash

1. Run the column clean method (Table4).


2. After the column wash, equilibrate the column for 3–5 min
   with the initial conditions for the acylation (Table5) or lacto-
   nization method (Table6).


3. After the sample run is complete, repeat the above steps before
   loading a new sample (seeNote 10).

Table 3

Methylthioadenosine nucleosidase assay setup

Component Volume (μl)

10 HEPES buffer 10

Water 40 x

10 KCl 10

5 μMMTAN 10

20 units/ml Xanthine oxidase 10

AHL synthase 10

Acyl-CoA/acyl-ACP x

10 SAM 10

Total 100

Table 4

Column clean HPLC method

Time (min) A%a B%b Flow rate (μl/min)

0.0 50.0 50.0 250.0

8.0 0.0 100.0 250.0

10.0 0.0 100.0 250.0

aA¼H 2 Oþ0.1% trifluoroacetic acid (TFA)

bB¼methanol

Table 5

Acylation HPLC methoda

Time (min) A%b B%c Flow rate (μl/min)

0.0 75.0 25.0 600.0

10.0 25.0 75.0 600.0

aObserve at 220 nm

bA¼H 2 Oþ0.1% trifluoroacetic acid (TFA)

cB¼acetonitrileþ0.1% TFA

Acyl-Homoserine Lactone Synthase Assays 171