Quorum Sensing

3.4.2 Standard Curve
for Lactonization Half-
Reaction Analytes

1. Prepare 1 mM MTA stock solution in nanopure water (seeNote
   11 ). Determine the concentration using MTA extinction coef-
   ficient at 275 nm,Δε 275 ¼14,900 M^1 cm^1.


2. Prepare two-fold serial dilutions of this sample, i.e., 500, 250,
   128, 64, 32, and 16μM.


3. Prepare standard solutions by adding 10 μl 6 M HCl (to
   simulate quenched reactions) to 260 μl water and 30μM
   MTA stock solution.


4. Inject the sample on to HPLC and run the lactonization HPLC
   method (Table6). Repeat this exercise at least three times
   (3 100 μl).


5. Determine peak area for MTA peak using an appropriate soft-
   ware for the LC instrument (Fig.5).


6. Plot peak area vs. MTA concentration and calculate the slope to
   determine the conversion factor for converting peak area to
   analyte concentration (seeNote 12).

3.4.3 Standard Curve
for Acylation Half-Reaction
Analytes

1. Prepare 2–3 mM stock solutions for apo-ACP, holo-ACP, and
   acyl-ACP in water (or buffer). Determine the carrier protein
   concentration using the extinction coefficient at 280 nm,
   Δε 280 ¼1490 M^1 cm^1.


2. Dilute this sample two-fold, at least six times as described in
   Subheading3.4.2,step 2.


3. Prepare standard solutions by mixing 260μl water, 30μl carrier
   protein, and 10 μl 2 M acetate buffer pH 4 (to simulate
   quenched reactions). Please note that acyl carrier proteins
   tend to precipitate out in HCl.


4. Run the sample using the acylation HPLC method (Table5)
   and determine the peak area for acyl-ACP and holo-ACP using
   an appropriate software that supports the LC instrument
   (Fig.5).


5. Plot peak area vs. acyl carrier protein concentration to generate
   the standard curve. The peak area must vary linearly with
   analyte concentration. The slope of this line is the conversion
   factor for determining concentration from peak areas in the
   chromatogram (seeNote 12).

Table 6

Lactonization HPLC methoda

Time (min) A%b B%c Flow rate (μl/min)

0.0 100.0 0.0 500.0

10.0 70.0 30.0 500.0

aObserve at 260 nm

bA¼H 2 Oþ0.1% trifluoroacetic acid (TFA)

cB¼acetonitrileþ0.1% TFA

172 Daniel Shin and Rajesh Nagarajan